Cytokine-based fusion proteins for treatment of multiple sclerosis

ABSTRACT

The present invention provides fusion proteins including an autoimmune antigen, an allergen antigen or an alloantigen, and an anti-inflammatory cytokine. Compositions and methods including the fusion proteins are also provided.

RELATED APPLICATION DATA

This application is a 37 U.S.C. §371 national phase application ofInternational Application No. PCT/US2007/022768, filed Oct. 29, 2007,which claims the benefit of U.S. Provisional Patent Application Ser. No.60/863,692, filed Oct. 31, 2006, the disclosures of each of which areincorporated herein by reference in their entireties.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 5218-156 FINAL_ST25.txt, 7,875 bytes in size, generatedon Oct. 6, 2014 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to compositions of matter and methods ofusing the same in regulating an immune response. The compositions ofmatter are useful in the treatment of immunological disorders, forexample, in the prevention and/or treatment of autoimmune disease,allergic disease, and transplant rejection.

BACKGROUND OF THE INVENTION

Autoimmune diseases are generally believed to be caused by the failureof the immune system to discriminate between antigens of foreigninvading organisms (non-self) and tissues native to the immune system'sown body (self). When this failure to discriminate between self andnon-self occurs, and the immune system reacts against self antigens, anautoimmune disorder may arise. Autoimmunity is a major cause of humandisease. Accordingly, new strategies are desired to elicitantigen-specific immunological tolerance as a means for treatment ofautoimmune disease (Fontoura et al. (2005) Int Rev Immunol 24, 415-446),and more generally, for the treatment of immunological disorders.

As opposed to generalized immunosuppression, antigen-specific regimensof tolerance induction can have improved efficacy because, at least inpart, anti-inflammatory activity would be focused on the smallpercentage of relevant pathogenic T cells minimizing the need for globalimmune suppression. Antigen-specific regimens can require temporaryrather than chronic administration, can be effective at lower dosesand/or can require local rather than systemic application, and thus, mayexhibit improved efficacy and cost-effectiveness with fewer adverse sideeffects. Thus, it is desirable to have improved therapeutic agents inthe treatment of immunological disorders.

SUMMARY OF THE INVENTION

Fusion proteins as described herein incorporating anti-inflammatorycytokines and immunodominant self antigen as separate domains of asingle protein may hold promise for development of antigen-specifictolerogenic vaccines. Proteins incorporating rat sequences of IL-1RA,IL-2, IL-4, IL-10 or IL-13 were expressed as fusion proteins containingthe major encephalitogenic region of myelin basic protein (MBP). In someembodiments, these fusion proteins were expressed via baculovirus (bv)expression systems and were shown to have cytokine-dependent andantigen-specific biological activity. In the case of the IL-2 and IL-4fusion proteins, covalent linkage of the cytokine in neuroantigendomains resulted in synergistic antigen presentation.

Fusion proteins as described herein may serve as antigen-specifictolerogens for treatment of autoimmune diseases. This novel concept forgeneration of tolerogenic vaccines was tested in some embodiments byconstructing fusion proteins including a tolerogenic or biasing cytokineand the major encephalitogenic peptide of guinea pig myelin basicprotein (GPMBP, i.e., neuroantigen or NAg). The cytokine domain waspredicted to condition antigen presenting cells (APC) whilesimultaneously targeting the covalently linked encephalitogenic peptideto the MHCII antigen processing pathway of those conditioned APC. Therank order of tolerogenic activity in the Lewis rat model of autoimmuneencephalomyelitis (EAE) was: NAgIL16>IL2NAg>IL1RA-NAg, IL13NAg≧IL10NAg,GPMBP, GP69-88, saline. NAgIL16 was also an effective inhibitor of EAEwhen administered after an encephalitogenic challenge during onset ofclinical signs.

The antigen-targeting activity of fusion proteins described herein suchas cytokine/NAg fusion proteins may be associated with mechanisms oftolerance induction. In some embodiments of the present invention,fusion proteins described herein were expressed including an N-terminalcytokine domain and a C-terminal neuroantigen (NAg) domain. In someembodiments, the cytokine domain included either rat IL-2 or IL-4, andthe NAg domain included the dominant encephalitogenic determinant ofguinea pig myelin basic protein (GPMBP). Subcutaneous administration ofIL2NAg (IL2-neuroantigen) into Lewis rats either before or after anencephalitogenic challenge resulted in an attenuated course of EAE. Incontrast, parallel treatment of rats with IL4NAg or NAg lackedtolerogenic activity. In the presence of IL-2R⁺ MHCII⁺ T cells, IL2NAgfusion proteins were at least 1,000 times more potent as an antigen thanNAg alone. In particular embodiments, the tolerogenic activity of IL2NAgin vivo and the enhanced potency in vitro were both dependent uponcovalent linkage of IL-2 and NAg. IL4NAg also exhibited enhancedantigenic potency. IL4NAg was approximately 100 fold more active thanNAg alone in the presence of splenic APC. According to some embodiments,the enhanced potency of IL4NAg was related to the covalent linkage ofcytokine and NAg and was blocked by soluble IL-4 or by a mAb specificfor IL-4. The activities of IL2NAg revealed a potential relationshipbetween NAg-targeting to activated T cells, T cell-mediated antigenpresentation, and tolerance induction.

Accordingly, embodiments of the present invention provide compositionsof matter for modulating an immune response and methods of modulating animmune response in a subject in need thereof.

Embodiments of the present invention further provide fusion proteinsincluding an autoimmune antigen, an allergen antigen or an alloantigenor a portion thereof, and an anti-inflammatory cytokine or a portionthereof.

Embodiments of the present invention provide methods of making a fusionprotein described herein.

Embodiments of the present invention provide an isolated nucleic acidencoding a fusion protein described herein as well as vectors includingthe isolated nucleic acids and hosts including the vectors.

Embodiments of the present invention provide compositions including atleast one fusion protein including an autoimmune antigen, an allergenantigen or an alloantigen or a portion thereof, an anti-inflammatorycytokine or a portion thereof and a pharmaceutically acceptable carrier,excipient or diluent.

Embodiments of the present invention provide methods of regulating animmunological disorder including administering an effective amount of atleast one fusion protein including an autoimmune antigen, an allergenantigen or an alloantigen or a portion thereof, and an anti-inflammatorycytokine or a portion thereof.

Embodiments of the present invention provide methods of modulating animmune response comprising administering at least one fusion protein inan amount sufficient to elicit a tolerogenic response, wherein the atleast one fusion protein includes an autoimmune antigen, an allergenantigen or an alloantigen or a portion thereof, and an anti-inflammatorycytokine or a portion thereof.

Embodiments of the present invention provide methods of modulatingantigen-presenting cell function including exposing anantigen-presenting cell to a fusion protein including an autoimmuneantigen, an allergen antigen or an alloantigen or a portion thereof, andan anti-inflammatory cytokine or a portion thereof.

Embodiments of the present invention provide kits including one or morecontainers having pharmaceutical dosage units including an effectiveamount of a fusion protein or a portion thereof as described herein,wherein the container is packaged with optional instructions for the usethereof.

Embodiments of the present invention provide uses of the fusion proteinsdescribed herein for the preparation of a medicament for carrying outthe utilities described herein.

Embodiments of the present invention further provide a novel approachfor the induction of antigen-specific tolerance, for example, fusionproteins incorporating anti-inflammatory or tolerogenic cytokines andthe dominant antigenic determinant of a self antigen (e.g. aneuroantigen (NAg) such as myelin basic protein (MBP)). The cytokine/NAgfusion proteins may target NAg to particular types of antigen-presentingcells (APC) by cytokine receptors present on the APC. The cytokinemoiety of the fusion protein may modulate APC function to engenderinhibitory or tolerogenic APC activities and also load the NAg intomajor histocompatibility complex (MHC) class II antigen processingpathways for presentation by the APC. NAg-specific T-helper cells thatrecognize NAg presented by the tolerogenic APC may be renderednonresponsive or may differentiate into regulatory T cells. Thisapproach may focus nonspecific inhibitory activities of particularcytokines onto rare NAg-specific T-helper cells that are responsible forCNS pathology.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in reference to the drawings and descriptionset forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The generation of constructs encoding rat cytokine/enterokinase(EK)/GP73-87/6his fusion proteins. Panel (A): Overlapping primers 5A01(SEQ ID NO:15) and 2F07 (SEQ ID NO:16) were extended to construct a DNAmolecule encoding the C-terminal enterokinase (EK)/GP73-87/6his peptide(SEQ ID NO:3). Panel (B): This DNA molecule was fused by overlapextension PCR to the coding sequence for rat IL1-RA, IL-2, IL-4, IL-10,or IL-13. In each case, the cytokine gene encoded the N-terminal domainand the EK/GP/6his DNA encoded the C-terminal domain. The strategy toconstruct the coding sequence for the IL2NAg (IL2.7) fusion protein isshown in FIG. 1B. The PCR product encoding rat IL-2 and the extensionproduct encoding the EK/GP73-87/6his tag were amplified in the presenceof excess upstream (3B12) and downstream (2F07) primers to generate theintact fusion sequence.

FIGS. 2A and 2B. IL1RANAg fusion proteins inhibited IL-1 activity. Lewisrat thymocytes (10⁶/well) were stimulated with 2.5 μg/ml Concanavalin Awith or without 20 ng/ml of human IL-1β in the presence or absence oftitrations (10%, 3.2%, 1%, or 0.3%; x-axis) of baculovirus supernatantscontaining the IL1RA/NAg4 fusion protein. Cells were pulsed with 1 μCiof [³H]thymidine during the last 24 hrs of a 3 day culture. Mean cpm areshown in FIG. 2A, whereas the same data is portrayed as percentinhibition in FIG. 2B. Percent inhibition of IL-1 stimulatedproliferation was defined as:

$\left\lbrack {1 - \frac{\left\lbrack {\left\lbrack {{{mean}\mspace{14mu}{{cpm}({IL1})}} - {{mean}\mspace{14mu}{{cpm}({control})}}} \right\rbrack{with}\mspace{14mu}{IL1RA}} \right\rbrack}{\left\lbrack {\left\lbrack {{{mean}\mspace{14mu}{{cpm}({IL1})}} - {{mean}\mspace{14mu}{{cpm}({control})}}} \right\rbrack{without}\mspace{14mu}{IL1RA}} \right\rbrack}} \right\rbrack*100{\%.}$Percent inhibition of Con A stimulated proliferation was defined as:

$\left\lbrack {1 - \frac{{mean}\mspace{14mu}{{cpm}\left( {{ConA}\mspace{14mu}{with}\mspace{14mu}{IL1RA}} \right)}}{{mean}\mspace{14mu}{{cpm}\left( {{ConA}\mspace{14mu}{without}\mspace{14mu}{IL1RA}} \right)}}} \right\rbrack*100{\%.}$

Values for mean cpm were obtained from triplicate cultures. These dataare representative of three separate experiments.

FIG. 3 (Panels A and B). The IL-2 fusion proteins presented IL-2bioactivity: Five baculovirus systems were tested for expression of IL-2bioactivity. Baculovirus supernatants containing the respective fusionproteins were titrated (10⁻¹ to 10⁻⁷; x-axis) and tested for IL-2bioactivity in the presence of CTLL indicator cells (10⁴/well). IL2Ekdeland IL2Ekdel2 represent sister baculovirus clones that express identicalproteins. These data are representative of three separate experiments.

FIG. 4 (Panels A and B). The IL4.4 (IL4/EK/GP73-87/6his) fusion proteinexhibited IL-4 bioactivity. Panel (A): Baculovirus expression systemsIL2.7, IL4.4, IL10.6, and IL13.6 were tested for expression of mitogenicactivity. Lewis rat thymocytes (5×10⁵/well) were cultured with 50 μg/mlPHA-P and 10 ng/ml IL-1β in the presence or absence of titrations (20%to 0.02%; i.e. 10^(−0.7) to 10^(−3.7); x-axis) of the respectivebaculovirus supernatant. (B) Designated baculovirus expression systemswere tested for IL-4 mitogenic activity. Thymocytes (10⁶/well) werecultured with 1 uM PMA and IL-2 (0.4% IL2 baculovirus supernatant) inthe presence or absence of titrations (10^(−0.7) to 10^(−3.7); x-axis)of the respective baculovirus supernatant. The IL2.7 baculovirussupernatant did not exhibit mitogenic activity in this assay becauseIL-2 was added in saturating concentrations to all wells. This bioassaywas therefore specific for IL-4. These data are representative of threeseparate experiments.

FIGS. 5A, 5B and 5C. IL4 and IL13 fusion proteins inhibitedγIFN-stimulated production of nitric oxide by macrophages. FIG. 5A: RatNR8383 macrophages (Helmke et al. (1987) In Vitro Cell Dev Biol 23,567-574; Patel et al. (1999) J Immunol 163, 5201-5210) (5×10⁴/well) werecultured with γIFN (100 U/ml), a fixed titration of the IL4.4 fusionprotein (0.5% titration of baculovirus supernatant), and designatedtitrations of IL13.6 baculovirus supernatant (x-axis). FIG. 5B:Alternatively, γIFN-stimulated NR8383 macrophages were cultured with afixed titration of the IL13.6 fusion protein (0.5% titration ofbaculovirus supernatant) and designated titrations of IL4.4 baculovirussupernatant (x-axis). FIG. 5C: NR8383 macrophages were cultured withγIFN (600 U/ml) and designated baculovirus supernatants (0.2%titration). These baculovirus supernatants were obtained from the 2^(nd)or 3^(rd) (P2 or P3) passage of the respective baculovirus in Sf9 insectcells. Nitric oxide production was measured by the accumulation ofnitrite by use of the Griess reagent (Ding et al., 1988). These data arerepresentative of three separate experiments.

FIGS. 6A, 6B, 6C and 6D. IL4 fusion proteins modulated T cell growth.The MBP-specific RsL.11 T cell clone (10⁵/well, FIG. 6A) or theconalbumin-specific Conal.8D9 clone (10⁵/well, FIG. 6B) were culturedwith or without 1% titrations of designated baculovirus supernatants.Cells were pulsed with [³H]thymidine during the last day of a 7-dayculture. FIG. 6C: Thymocytes (10⁶/well) were cultured with 1 uM PMA andIL-2 (0.4% IL2 baculovirus supernatant) in the presence or absence oftitrations (10⁻¹ to 10⁻⁴; x-axis) of the respective baculovirussupernatant. Cells were pulsed with [³H]thymidine during the last day ofa 3-day culture. FIG. 6D: NR8383 macrophages were cultured withgamma-interferon (100 U/ml) in the presence or absence of designatedbaculovirus titrations. After 3 days of culture, supernatants werecollected and assayed for evidence of nitric oxide production by theGriess reaction. These data are representative of three separateexperiments.

FIGS. 7A and 7B. The IL10.6 fusion protein enhanced IL-2R expression andIL-2 dependent T cell growth. FIG. 7A: MBP-specific RsL.11 T cells(25,000/well) were cultured with or without 10 pM, 100 pM, or 1 nM IL-2and designated concentrations of purified IL10.6 (100 fM to 100 nM;x-axis). IL10.6 represents the IL10/Ek/NAg/6his fusion protein. Cellswere pulsed with [³H]thymidine during the last day of a 4-day culture.FIG. 7B: RsL.11 T cells (5×10⁵/well; 24-well plate) were cultured withIL-2 in the presence or absence of a 1% titration of designatedbaculovirus supernatants for 3 days. Cells were then analyzed by flowcytometry for cell size (left panels) or expression of the OX39 IL-2R(IL-2 receptor) marker (right panels). The OX6 IgG1 mAb exhibitedstaining equivalent to cells stained without primary Ab and was used asan isotype control. These data are representative of three separateexperiments.

FIG. 8. SDS-PAGE analysis of purified cytokine/NAg fusion proteins.Proteins eluted from the Ni resin were analyzed on 12% SDS-PAGE gels.Lane 1, IL1RA/NAg with a predicted MW of 20,609 daltons and onepotential N-linked site. Lane 2, IL2Ekdel with a predicted MW of 17,996daltons and no potential N-linked sites. Lane 3, IL2-D with a predictedMW of 16,316 daltons and no potential N-linked sites. Lane 4, IL4Ekdelwith a predicted MW of 16,654 daltons and four potential N-linked sites.Lane 5, IL10.6 with a predicted MW of 21,744 daltons and one potentialN-linked site. Lane 6, IL13.6 with a predicted MW of 15,466 daltons andfour potential N-linked sites. The image for each lane was obtained fromindependently performed SDS-PAGE analyses. These data are representativeof three separate experiments.

FIGS. 9A and 9B. Purified cytokine/NAg fusion proteins stimulatedantigen-specific proliferation of MBP-specific T cells. FIG. 9A: RsL.11T cells (2.5×10⁴/well) and irradiated splenocytes (5×10⁵/well) werecultured with designated concentrations of cytokine/NAg fusion proteinor GPMBP in the presence or absence of a mAb (OX6) specific for rat MHCclass II RT1B glycoproteins. In the presence of OX6, T cells culturedwith 100 fM-10 nM cytokine/NAg exhibited <1,000 cpm, and T cellscultured with 100 nM cytokine/NAg exhibited <10,000 cpm of [³H]thymidineincorporation. FIG. 9B: GPMBP-sensitized draining lymph node cells (LNC)were obtained 60 days after sensitization with DHFR/NAg in CFA. LNC(5×10⁵/well) were cultured with 100 nM cytokine/NAg or GPMBP in thepresence or absence of OX6. Cells were pulsed with [³H]thymidine duringthe last 24 hrs of a 3-day culture. These data are representative ofthree separate experiments.

FIGS. 10A and 10B. Cytokine/NAg fusion proteins elicitedantigen-specific killing during MHCII-dependent antigen presentation.FIG. 10A: MHCII⁺ R1-trans T cells (2.5×10⁴/well) and irradiated RsL.11 Tcells (1000 rads) were cultured with IL-2 (0.4% v/v IL2-D baculovirussupernatant) in the presence or absence of 1 uM GPMBP and designatedtitrations of OX6 (x-axis). FIG. 10B: MHCII⁺ R1-trans T cells werestarved of IL-2 for 24 hrs, were washed, and were cultured withdesignated concentrations of purified cytokine/NAg. After 24 hrs ofculture, irradiated RsL.11 T cells (2.5×10⁴/well) and IL-2 were added toeach well. Cultures were pulsed with [³H]thymidine during the last 24hrs of a 3-day culture. These data are representative of three separateexperiments.

FIG. 11. Time course of EAE induction and relapse. FIG. 11 presents agraphical depiction of data shown in Experiment #2 of Table 6.

FIGS. 12A and 12B. Purified cytokine/NAg fusion proteins stimulated theantigenic proliferation of an MBP-specific T cell clone. FIG. 12A:RsL.11 T cells (25,000/well) and irradiated splenocytes (500,000/well)were cultured with designated concentrations of the respective fusionprotein. FIG. 12B: RsL.11 T cells and irradiated splenic APC werecultured with designated concentrations of GPMBP, IL4.4, or IL2.7 in thepresence or absence of the anti-rat I-A MHCII OX6 mAb or anti-rat I-EMHCII OX17 mAb. Cultures were pulsed with [³H]thymidine during the last24 hrs of a 72 hr culture. These data are representative of threeexperiments.

FIGS. 13A, 13B, 13C and 13D. Enhanced potency of IL4.4 and IL2.7 wasdependent upon the covalent linkage of cytokine and NAg. FIGS. 13A and13B: RsL.11 T cells (25,000/well) and irradiated splenocytes(500,000/well) were used to assay antigenic activity of the NAg. FIG.13C: Thymocytes (1×10⁶/well) cultured with 1 uM PMA and rat IL-2 (0.4%baculovirus supernatant) were used to assay IL-4 activity. FIG. 13D:CTLL cells (1×10⁴/well) were used to assay IL-2 activity. (FIGS. 13A-D)These cells were cultured with or without designated concentrations ofGPMBP, IL4.4, or IL2.7 in the presence or absence of IL-4 or IL-2 (1% or0.1% baculovirus supernatants). Cultures were pulsed with [³H]thymidineduring the last 24 hrs of a 72 hr culture. These data are representativeof three experiments.

FIG. 14. T cell-mediated presentation of IL2.7 was inhibited by solubleIL-2. The MHCII⁺ blastogenic R1-trans T cell clone was starved of IL-2for 24 hours before the assay. At the initiation of the assay, R1-transT cells were cultured with irradiated MBP-specific RsL.11 responders anddesignated concentrations of IL2.7 or GPMBP (x-axis). Rat IL-2 (0.4% ofa baculovirus supernatant) was added at 0, 4, or 24 hours afterinitiation of culture. Cultures were pulsed with [³H]thymidine duringthe last 24 hrs of culture of a 72 hr assay. These data arerepresentative of three experiments.

FIGS. 15A and 15B. In the presence of irradiated splenic APC,presentation of IL2.7 and IL4.4 were inhibited by IL-2 and IL-4,respectively. RsL.11 T cells and irradiated splenic APC were culturedfor 3 days with designated concentrations of GPMBP, IL4.4, or IL2.7 inthe presence or absence of 1% v/v baculovirus supernatant containingIL-2 or IL-4. IL-2 or IL-4 was added to culture 3 hr before antigen.Cultures were pulsed with [³H]thymidine during the last 24 hrs of a 72hr culture. These data are representative of three experiments.

FIG. 16. IL4.4 and MBP are equally accessible to antigen processing.RsL.11 T cells and irradiated splenic APC were cultured for 3 days withdesignated concentrations of GPMBP, IL4.4, or IL2.7 in the presence orabsence of the OX81 mAb against rat IL-4. The mAb were added to culture3 hr before antigen. Cultures were pulsed with [³H]thymidine during thelast 24 hrs of a 72 hr culture. These data are representative of threeexperiments.

FIG. 17. The time course of the mean severity of experimental autoimmuneencephalomyelitis (EAE) based on the data presented in Table 12.

FIG. 18. Higher doses of NAgIL-16 L induced more efficient tolerance.Rats were injected with of designated doses NAgIL16 on days −21, −14,and −7 (n=4 for each group). Rats were then challenged with 50 μgDHFR-NAg in CFA on day 0. The mean cumulative score (p<0.01, p<0.01,p<0.05, p<0.05), the mean maximal intensity (p<0.01, p<0.01, ns, ns),and the mean day of onset (p<0.001; all groups) of rats treated with0.5, 1.0, 2.5, or 5.0 nmoles NAgIL16-L differed significantly from thecontrol group (saline), respectively.

FIG. 19. NAgIL16 was targeted for presentation by nonadherent ConA-activated splenic T cells

FIG. 20. Biological activity of IFNβNAg and IL2NAg fusion proteins.

FIGS. 21A and 21B. Administration of NAgIL16 during clinical diseasehalts EAE progression and prevents subsequent relapse. Data presented intabular form (Table 19, Experiment 2) are shown as a time course of EAE.On day 11, rats were matched for clinical signs of EAE and were randomlyassigned to one of two groups that were injected with either NAg(GP69-88) or NAgIL16L. (FIG. 21A) Treatments were on day 11 (5 nmoles insaline i.v.), day 12 (5 nmoles in saline i.p.), and day 14 (2 nmoles insaline i.v.) (see arrows). The cumulative EAE severity per day forNAgIL16-treated rats was significantly less than that for NAg-treatedrats on days 13, 13.5, and 14 (p=0.013, 0.005, and 0.009, respectively).The mean cumulative scores (tallied after the first treatment on day 11)also were significantly different (p<0.001) (Mann-Whitney Test). (FIG.21B) The frequency of relapses (onset after day 21) for NAg-treated rats(6 of 7, 85.7%) was significantly higher than for NAgIL16-treated rats(0 of 7) (p=0.0047, Fisher's Exact Test).

DETAILED DESCRIPTION

The present invention will now be described with reference to thefollowing embodiments. As is apparent by these descriptions, thisinvention can be embodied in different forms and should not be construedas limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Except as otherwise indicated, standard methods can be used for theproduction of viral and non-viral vectors, manipulation of nucleic acidsequences, production of transformed cells, and the like according tothe present invention. Such techniques are known to those skilled in theart. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al. CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc., New York).

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

1. Definitions

As used herein, “a” or “an” or “the” can mean one or more than one. Alsoas used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount.

The term “modulate,” “modulates” or “modulation” refers to enhancement(e.g., an increase) or inhibition (e.g., a reduction) in the specifiedactivity.

The term “regulate” as used herein refers to the ability to affect amethod, process, state of being, disorder or the like. The effect may bethat of prevention, treatment or modulation.

By the terms “treat,” “treating” or “treatment of,” it is intended thatthe severity of the disorder or the symptoms of the disorder arereduced, or the disorder is partially or entirely eliminated, ascompared to that which would occur in the absence of treatment.Treatment does not require the achievement of a complete cure of thedisorder.

By the terms “preventing” or “prevention”, it is intended that theinventive methods eliminate or reduce the incidence or onset of thedisorder, as compared to that which would occur in the absence of themeasure taken. Alternatively stated, the present methods slow, delay,control, or decrease the likelihood or probability of the disorder inthe subject, as compared to that which would occur in the absence of themeasure taken.

A “therapeutically effective” or “effective” amount is intended todesignate a dose that causes a relief of symptoms of a disease ordisorder as noted through clinical testing and evaluation, patientobservation, and/or the like. “Effective amount” or “effective” canfurther designate a dose that causes a detectable change in biologicalor chemical activity. The detectable changes may be detected and/orfurther quantified by one skilled in the art for the relevant mechanismor process. Moreover, “effective amount” or “effective” can designate anamount that maintains a desired physiological state, i.e., reduces orprevents significant decline and/or promotes improvement in thecondition of interest. As is generally understood in the art, the dosagewill vary depending on the administration routes, symptoms and bodyweight of the patient but also depending upon the compound beingadministered.

“Immune response” generally refers to innate and acquired immuneresponses including, but not limited to, both humoral immune responses(mediated by B lymphocytes) and cellular immune responses (mediated by Tlymphocytes). An immune response may be beneficial and lead to immunityagainst infectious pathogens, or an immune response may be pathogenicand lead to autoimmune or hypersensitivity disease. Immune responsesagainst foreign viruses, bacteria, fungi, parasites typically representbeneficial adaptive immune responses. Immune responses against selftissues, innocuous foreign objects (e.g., dust mite or pollen allergens,etc.), or tissue transplants represent examples of adverse maladaptiveimmune responses.

The term “antigen” as used herein means a substance or compound thatstimulates an immune response. Although usually a protein orpolysaccharide, antigens may be any type of molecule, which can includesmall molecules (haptens) that are coupled to a carrier-protein.

By the term “immunogenic” it is meant any substance or compound thatstimulates an immune response.

By the term “tolerogen” it is meant any substance that stimulatesimmunological tolerance. By the terms “tolerogenic” or “tolerogenicactivity” it is meant that a response of immunological tolerance isinduced by an antigen or antigenic substance or an activity that resultsin the induction of immunological tolerance toward an antigen orantigenic substance.

The term “tolerance” as used herein refers to a decreased level of animmune response, a delay in the onset or progression of an immuneresponse and/or a reduced risk of the onset or progression of an immuneresponse. “Specific” immunological tolerance occurs when immunologicaltolerance is preferentially invoked against certain antigens incomparison with others. “Active” immunological tolerance refers to astate in which the tolerance effect(s) are the result of an ongoingbiological process: for example, down-regulation of specific effectorcells by suppressor cells. “Sustained tolerance” is tolerance thatmeasurably persists for an extended period of time.

The terms “vaccination” or “immunization” are well-understood in theart. For example, the terms vaccination or immunization can beunderstood to be a process that increases a subject's immune reaction toantigen and therefore the ability to resist or overcome infection. Inthe case of the present invention, vaccination or immunization maydecrease the recipient's immune response against self antigens therebydecreasing the likelihood of an autoimmune response.

“Polypeptide” as used herein, is used interchangeably with “protein,”and refers to a polymer of amino acids (dipeptide or greater) linkedthrough peptide bonds. Thus, the term “polypeptide” includes proteins,oligopeptides, protein fragments, protein analogs and the like. The term“polypeptide” contemplates polypeptides as defined above that areencoded by nucleic acids, are recombinantly produced, are isolated froman appropriate source, or are synthesized.

As used herein, a “functional” polypeptide is one that retains at leastone biological activity normally associated with that polypeptide.Preferably, a “functional” polypeptide retains all of the activitiespossessed by the unmodified peptide. By “retains” biological activity,it is meant that the polypeptide retains at least about 50%, 60%, 75%,85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of thenative polypeptide (and can even have a higher level of activity thanthe native polypeptide). A “non-functional” polypeptide is one thatexhibits essentially no detectable biological activity normallyassociated with the polypeptide (e.g., at most, only an insignificantamount, e.g., less than about 10% or even 5%).

“Fusion protein” as used herein, refers to a protein produced when twoheterologous nucleotide sequences or fragments thereof coding for two(or more) different polypeptides, or fragments thereof, are fusedtogether in the correct translational reading frame. The two or moredifferent polypeptides, or fragments thereof, include those not foundfused together in nature and/or include naturally occurring mutants.

As used herein, a “fragment” is one that substantially retains at leastone biological activity normally associated with that protein orpolypeptide. In particular embodiments, the “fragment” substantiallyretains all of the activities possessed by the unmodified protein. By“substantially retains” biological activity, it is meant that theprotein retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%,99%, or more, of the biological activity of the native protein (and caneven have a higher level of activity than the native protein).

A “recombinant” nucleic acid is one that has been created using geneticengineering techniques.

A “recombinant polypeptide” is one that is produced from a recombinantnucleic acid.

As used herein, an “isolated” nucleic acid (e.g., an “isolated DNA” oran “isolated vector genome”) means a nucleic acid separated orsubstantially free from at least some of the other components of thenaturally occurring organism or virus, such as for example, the cell orviral structural components or other polypeptides or nucleic acidscommonly found associated with the nucleic acid.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide. As used herein,the “isolated” polypeptide is at least about 25%, 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99% or more pure (w/w).

A “heterologous nucleotide sequence” will typically be a sequence thatis not naturally-occurring in the vector. Alternatively, a heterologousnucleotide sequence can refer to a sequence that is placed into anon-naturally occurring environment (e.g., by association with apromoter with which it is not naturally associated; in a cell that doesnot contain an endogenous form of the heterologous nucleotide sequenceand/or under the direction of a promoter and/or other regulatoryelements with which it is not normally associate, in a cell that doescontain an endogenous form of the heterologous nucleotide sequence.).

There are no particular limits to the size of the heterologous nucleicacid. In particular embodiments, the heterologous nucleic acid is atleast about 15, 18, 24, 50, 100, 250, 500, 1000, 1500, 2000, 3000, 4000or more nucleotides long and/or less than about 4000, 3000, 2000, 1500,1000, 500, 250 or 100 nucleotides long.

As used herein, a “vector” or “delivery vector” can be a viral ornon-viral vector that is used to deliver a nucleic acid to a cell,tissue or subject.

A “recombinant” vector or delivery vector refers to a viral or non-viralvector that comprises one or more heterologous nucleotide sequences(i.e., transgenes), e.g., two, three, four, five or more heterologousnucleotide sequences. In an embodiment of the invention, the recombinantvectors and delivery vectors of the invention encode a fusionpolypeptide of NAg and cytokines such as IL-2, IL-16 or IFN-β, but canalso include one or more additional heterologous sequences, for example,sequences encoding C- or N-terminal modifications and linker moieties.

As used herein, the term “viral vector” or “viral delivery vector” canrefer to a virus particle that functions as a nucleic acid deliveryvehicle, and which comprises the vector genome packaged within a virion.Alternatively, these terms can be used to refer to the vector genomewhen used as a nucleic acid delivery vehicle in the absence of thevirion.

A viral “vector genome” refers to the viral genomic DNA or RNA, ineither its naturally occurring or modified form. A “recombinant vectorgenome” is a viral genome (e.g., vDNA) that comprises one or moreheterologous nucleotide sequence(s).

As used herein, the term “host cell” comprises prokaryotic cells andeukaryotic cells. Exemplary prokaryotic host cells include E. coli,Bacillus subtilis, etc. Exemplary eukaryotic cells include yeast cells,insect cells, mammal cells, etc.

2. Active Agents

Embodiments of the present invention provide a fusion proteincomprising, consisting essentially of or consisting of an autoimmuneantigen or a portion thereof, and an anti-inflammatory cytokine orportion thereof. Embodiments of the present invention provide a fusionprotein comprising, consisting essentially of or consisting of anallergen antigen (for example, antigens derived from common allergensthat cause allergic hypersensitivity disease) or a portion thereof, andan anti-inflammatory cytokine or portion thereof. Embodiments of thepresent invention provide a fusion protein comprising, consistingessentially of or consisting of an alloantigen (e.g. an allogeneictissue transplantation antigen) or a portion thereof, and ananti-inflammatory cytokine or portion thereof.

Autoimmune antigens of the present invention include neuroantigens (NAg)derived from the central or peripheral nervous system. In someembodiments, the neuroantigen is myelin basic protein (MBP) or a portionthereof. In other embodiments, the neuroantigen is proteolipid protein(PLP), myelin oligodendrocyte glycoprotein (MOG), myelin-associatedoligodendrocytic basic protein, or other nervous system-derived proteinsor a portion thereof. In some embodiments, the autoimmune antigenincludes an encephalitogenic determinant of the myelin basic protein ora portion thereof. In other embodiments, the encephalitogenicdeterminant of the myelin basic protein includes amino acids of SEQ IDNO. 2. In general, the encephalitogenic determinant of neuroantigensinclude amino acid sequences of any self protein that may become thetarget of an encephalitogenic autoimmune attack. Further, as understoodby one skilled in the art, the location of the encephalitogenicdeterminant can, in part, be determined by the highly polymorphicpeptide binding properties of MHC class II glycoproteins.

Autoimmune antigens also include self-antigens such as insulin(autoimmune diabetes), the thyroid-stimulating hormone receptor (Grave'sdisease), platelets (thrombocytopenic purpura), neuromuscular junction(myasthenia gravis), red blood cells (autoimmune hemolytic anemia),intracellular antigens (spliceosomes, ribosomes, nucleic acid, etc insystemic lupus erythematosus). Autoimmune antigens can include any selfmolecule including protein, carbohydrate, lipid, or nucleic acid or anycombination thereof that is made normally within the body that wouldconstitute a part of the body that may become targeted in a particularautoimmune disease.

The anti-inflammatory cytokine is a naturally occurring or recombinantprotein, analog thereof or fragment thereof that elicits ananti-inflammatory response in a cell that has a receptor for thatcytokine. Cytokines of the present invention can include interleukinreceptor antagonists from any species including murine and human such asIL-1-RA. Cytokines of the present invention can further includeinterleukins from any species including murine and human such as IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17 and IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25,IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-31, IL-32, and IL-33,hematopoietic factors such as macrophage colony-stimulating factor(M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF) and erythropoietin, tumornecrosis factors (TNF) such as TNF-α and TGF-β, lymphokines such aslymphotoxin, regulators of metabolic processes such as leptin,interferons such as IFN-α, IFN-β, and IFN-γ and chemokines. In someembodiments of the invention, the cytokine is an interleukin such asIL-2, IL-16 or IFN-β. One or more of the fusion proteins of the presentinvention can display at least some cytokine biological activity.

According to embodiments of the present invention, the autoimmuneantigen, the allergen antigen or the alloantigen and theanti-inflammatory cytokine are covalently linked. In some embodiments,the autoimmune antigen, the allergen antigen or the alloantigen and theanti-inflammatory cytokine are directly linked. In other embodiments,the autoimmune antigen, the allergen antigen or the alloantigen and theanti-inflammatory cytokine are linked through a linking moiety. Thelinking moiety can be an amino acid or peptide moiety. In someembodiments, the linking moiety is a cleavable linker. In particularembodiments, the linking moiety is an enterokinase domain.

Embodiments of the present invention further provide an isolated nucleicacid (e.g., an “isolated DNA” or an “isolated vector genome”) thatencodes the fusion protein described herein. The nucleic acid isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, such as forexample, the cell or viral structural components or other polypeptidesor nucleic acids commonly found associated with the nucleic acid. Thecoding sequence for a polypeptide constituting the active agents of thepresent invention is transcribed, and optionally, translated. Accordingto embodiments of the present invention, transcription and translationof the coding sequence will result in production of a fusion proteindescribed. In some embodiments, the isolated nucleic acid encodes acytokine, an interleukin receptor antagonist, an interleukin and/or alinker moiety to provide a fusion protein including a cytokine and aninterleukin receptor antagonist or interleukin. In particularembodiments, the isolated nucleic acid encodes a fusion proteinincluding NAg and IL-2, IL-16 or IFN-β as examples.

It will be appreciated by those skilled in the art that there can bevariability in the nucleic acids that encode the fusion polypeptides ofthe present invention due to the degeneracy of the genetic code. Thedegeneracy of the genetic code, which allows different nucleic acidsequences to code for the same polypeptide, is well known in theliterature (see Table 1).

Further variation in the nucleic acid sequence can be introduced by thepresence (or absence) of non-translated sequences, such as intronicsequences and 5′ and 3′ untranslated sequences.

Moreover, the isolated nucleic acids of the invention encompass thosenucleic acids encoding fusion proteins that have at least about 60%,70%, 80%, 90%, 95%, 97%, 98% or higher amino acid sequence similaritywith the polypeptide sequences specifically disclosed herein or to thoseknown sequences corresponding to proteins included in aspects of thepresent invention (or fragments thereof) and further encode functionalfusion proteins as defined herein.

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or polypeptide has sequence identity orsimilarity to a known sequence. Sequence identity and/or similarity canbe determined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith &Waterman (1981), Adv. Appl. Math. 2, 482, by the sequence identityalignment algorithm of Needleman & Wunsch (1970), J. Mol. Biol. 48, 443,by the search for similarity method of Pearson & Lipman (1988), Proc.Natl. Acad. Sci. USA 85, 2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al.(1984), Nucl. Acid Res. 12, 387-395, preferably using the defaultsettings, or by inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, (1987) J. Mol.Evol. 35, 351-360; the method is similar to that described by Higgins &Sharp (1989), CABIOS 5, 151-153.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al. (1990), J. Mol. Biol. 215, 403-410, and Karlin et al.(1993), Proc. Natl. Acad. Sci. USA 90, 5873-5787. A particularly usefulBLAST program is the WU-BLAST-2 program which was obtained from Altschulet al. (1996), Methods in Enzymology, 266, 460-480. WU-BLAST-2 usesseveral search parameters, which are preferably set to the defaultvalues. The parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values can be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., (1997) Nucleic Acids Res. 25, 3389-3402.

A percentage amino acid sequence identity value can be determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

The alignment can include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the polypeptides specifically disclosed herein,it is understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than a sequence specificallydisclosed herein, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

To modify the amino acid sequences of the fusion proteins of the presentinvention, amino acid substitutions can be based on any characteristicknown in the art, including the relative similarity or differences ofthe amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Inparticular embodiments, conservative substitutions (i.e., substitutionwith an amino acid residue having similar properties) are made in theamino acid sequence encoding a polypeptide of the invention.

In making amino acid substitutions, the hydropathic index of amino acidscan be considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (see, Kyte and Doolittle (1982), J. Mol. Biol.157:105; incorporated herein by reference in its entirety). It isaccepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle, Id.),and these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine. (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is also understood in the art that the substitution of amino acidscan be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(incorporated herein by reference in its entirety) states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±I); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Isolated nucleic acids of this invention include RNA, DNA (includingcDNAs) and chimeras thereof. The isolated nucleic acids can furthercomprise modified nucleotides or nucleotide analogs.

The isolated nucleic acids encoding the polypeptides of the inventioncan be associated with appropriate expression control sequences, e.g.,transcription/translation control signals and polyadenylation signals.

It will be appreciated that a variety of promoter/enhancer elements canbe used depending on the level and tissue-specific expression desired.The promoter can be constitutive or inducible (e.g., the metalothioneinpromoter or a hormone inducible promoter), depending on the pattern ofexpression desired. The promoter can be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced. Thepromoter is chosen so that it will function in the target cell(s) ofinterest.

The present invention further provides methods of making fusion proteinsdescribed herein. Methods of making fusion proteins are well understoodin the art. According to embodiments of the present invention, methodsof making fusion proteins include those in accordance with U.S. Pat.Nos. 4,701,416; 5,496,924; 5,521,288; 5,837,816; 5,981,221; 5,994,104;6,109,885; 6,211,342; 6,211,427; 6,369,199; 6,482,409; 6,555,342;6,972,322; 6,987,006 7,087,411 and 7,112,659 incorporated herein byreference in their entirety. Such methods include growing a host cellincluding a vector that includes nucleic acids encoding the fusionprotein under conditions appropriate for expression and subsequentisolation of the fusion protein. Accordingly, the isolated nucleic acidsencoding a polypeptide constituting the fusion protein of the inventioncan be incorporated into a vector, e.g., for the purposes of cloning orother laboratory manipulations, recombinant protein production, or genedelivery. Exemplary vectors include bacterial artificial chromosomes,cosmids, yeast artificial chromosomes, phage, plasmids, lipid vectorsand viral vectors (described in more detail below).

In particular embodiments, the isolated nucleic acid is incorporatedinto an expression vector. In further embodiments of the presentinvention, the vector including the isolated nucleic acids describedherein are included in a host cell. Expression vectors compatible withvarious host cells are well known in the art and contain suitableelements for transcription and translation of nucleic acids. Typically,an expression vector contains an “expression cassette,” which includes,in the 5′ to 3′ direction, a promoter, a coding sequence encoding apolypeptide of the invention or active fragment thereof operativelyassociated with the promoter, and, optionally, a termination sequenceincluding a stop signal for RNA polymerase and a polyadenylation signalfor polyadenylase.

Expression vectors can be designed for expression of polypeptides inprokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g., in thebaculovirus expression system), yeast cells or mammalian cells. Somesuitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSecl (Baldari et al., (1987) EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,(1987) Gene 54:113-123), and pYES2 (Invitrogen™, San Diego, Calif.).Baculovirus vectors available for expression of nucleic acids to produceproteins in cultured insect cells (e.g., Sf 9 cells) include theBac-to-Bac® Baculovirus Expression System from Invitrogen.

Examples of mammalian expression vectors include pCDM8 (Seed, (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, adenovirus 2, cytomegalovirusand Simian Virus 40.

In addition to the regulatory control sequences discussed above, therecombinant expression vector can contain additional nucleotidesequences. For example, the recombinant expression vector can encode aselectable marker gene to identify host cells that have incorporated thevector and/or may comprise another heterologous sequence of interest.

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acids (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNAcomplexes, cell sonication, gene bombardment using high velocitymicroprojectiles, and viral-mediated transfection. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory manuals.

3. Formulations and Administration

Embodiments of the present invention provide a composition including anautoimmune antigen, an allergen antigen or an alloantigen or a portionthereof and an anti-inflammatory cytokine or portion thereof, and apharmaceutically acceptable carrier, excipient or diluent. In someembodiments, the composition includes at least two fusion proteins. Insuch embodiments, at least one fusion protein includes ananti-inflammatory cytokine or portion thereof that is different from theanti-inflammatory cytokine or portion thereof of at least one otherfusion protein.

In terms of administration, the most suitable route in any given casewill depend on the nature and severity of the condition being treatedand on the fusion protein, viral vector, nucleic acid or pharmaceuticalformulation being administered.

The fusion proteins, viral vectors and nucleic acids (e.g., DNA and/orRNA) of the invention can be formulated for administration in apharmaceutical carrier in accordance with known techniques. See, e.g.,Remington, The Science And Practice of Pharmacy (9th Ed. 1995). In themanufacture of a pharmaceutical formulation according to the invention,the fusion protein, viral vector or nucleic acid is typically admixedwith, inter alia, an acceptable carrier. The carrier can be a solid or aliquid, or both, and is optionally formulated as a unit-doseformulation, which can be prepared by any of the well-known techniquesof pharmacy.

The carriers and additives used for such pharmaceutical compositions cantake a variety of forms depending on the anticipated mode ofadministration. Thus, compositions for oral administration may be, forexample, solid preparations such as tablets, sugar-coated tablets, hardcapsules, soft capsules, granules, powders and the like, with suitablecarriers and additives being starches, sugars, binders, diluents,granulating agents, lubricants, disintegrating agents and the like.Because of their ease of use and higher patient compliance, tablets andcapsules represent the most advantageous oral dosage forms for manymedical conditions.

Similarly, compositions for liquid preparations include solutions,emulsions, dispersions, suspensions, syrups, elixirs, and the like withsuitable carriers and additives being water, alcohols, oils, glycols,preservatives, flavoring agents, coloring agents, suspending agents, andthe like.

In the case of a solution, it can be lyophilized to a powder and thenreconstituted immediately prior to use. For dispersions and suspensions,appropriate carriers and additives include aqueous gums, celluloses,silicates or oils.

For injection, the carrier is typically a liquid, such as sterilepyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.),parenterally acceptable oil including polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil, with other additivesfor aiding solubility or preservation may also be included. For othermethods of administration, the carrier can be either solid or liquid.

For oral administration, the fusion protein, viral vector or nucleicacid can be administered in solid dosage forms, such as capsules,tablets, and powders, or in liquid dosage forms, such as elixirs,syrups, and suspensions. The fusion protein, viral vector or nucleicacid can be encapsulated in gelatin capsules together with inactiveingredients and powdered carriers, such as glucose, lactose, sucrose,mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that can beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

Formulations suitable for buccal (sub-lingual) administration includelozenges including the fusion protein, viral vector or nucleic acid in aflavored base, usually sucrose and acacia or tragacanth; and pastillesincluding the fusion protein, viral vector or nucleic acid in an inertbase such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration can include sterile aqueous and non-aqueous injectionsolutions of the fusion protein, viral vector or nucleic acid, whichpreparations are generally isotonic with the blood of the intendedrecipient. These preparations can contain anti-oxidants, buffers,bacteriostats and solutes, which render the formulation isotonic withthe blood of the intended recipient. Aqueous and non-aqueous sterilesuspensions can include suspending agents and thickening agents. Theformulations can be presented in unit\dose or multi-dose containers, forexample sealed ampoules and vials, and can be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or water-for-injection immediatelyprior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets. For example, in one aspect of thepresent invention, there is provided an injectable, stable, sterilecomposition including a fusion protein, viral vector or nucleic acid ofthe invention, in a unit dosage form in a sealed container. Optionally,the composition is provided in the form of a lyophilizate, which iscapable of being reconstituted with a suitable pharmaceuticallyacceptable carrier to form a liquid composition suitable for injectionthereof into a subject.

Formulations suitable for rectal or vaginal administration can bepresented as suppositories. These can be prepared by admixing the fusionprotein, viral vector or nucleic acid with one or more conventionalexcipients or carriers, for example, cocoa butter, polyethylene glycolor a suppository wax, which are solid at room temperature, but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the fusion protein, viral vector or nucleic acid.

Formulations suitable for topical application to the skin can take theform of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.Carriers that can be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration can also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6):318(1986)) and typically take the form of an optionally buffered aqueoussolution. Suitable formulations comprise citrate or bis\tris buffer (pH6) or ethanol/water.

The fusion protein, viral vector or nucleic acid can be formulated fornasal administration or otherwise administered to the lungs of a subjectby any suitable means, for example, by an aerosol suspension ofrespirable particles including the fusion protein, viral vector ornucleic acid, which the subject inhales. The respirable particles can beliquid or solid. The term “aerosol” includes any gas-borne suspendedphase, which is capable of being inhaled into the bronchioles or nasalpassages. Specifically, aerosol includes a gas-borne suspension ofdroplets, as can be produced in a metered dose inhaler or nebulizer, orin a mist sprayer. Aerosol also includes a dry powder compositionsuspended in air or other carrier gas, which can be delivered byinsufflation from an inhaler device, for example. See Ganderton & Jones,Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda(1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313;and Raebum et al. (1992) J. Pharmacol. Toxicol. Methods 27:143-159.Aerosols of liquid particles can be produced by any suitable means, suchas with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer,as is known to those of skill in the art. See, e.g., U.S. Pat. No.4,501,729. Aerosols of solid particles including the fusion protein,viral vector or nucleic acid can likewise be produced with any solidparticulate medicament aerosol generator, by techniques known in thepharmaceutical art.

Alternatively, one can administer the fusion protein, viral vector ornucleic acid in a local rather than systemic manner, for example, in adepot or sustained-release formulation.

In particular embodiments of the invention, administration is bysubcutaneous or intradermal administration. Subcutaneous and intradermaladministration can be by any method known in the art including, but notlimited to, injection, gene gun, powderject device, bioject device,microenhancer array, microneedles, and scarification (i.e., abrading thesurface and then applying a solution including the fusion protein, viralvector or nucleic acid).

In other embodiments, the fusion protein, viral vector or nucleic acidis administered intramuscularly, for example, by intramuscular injectionor by local administration.

Nucleic acids (e.g., DNA and/or RNA) can also be delivered inassociation with liposomes, such as lecithin liposomes or otherliposomes known in the art (for example, as described in WO 93/24640)and may further be associated with an adjuvant. Liposomes includingcationic lipids interact spontaneously and rapidly with polyanions, suchas DNA and RNA, resulting in liposome/nucleic acid complexes thatcapture up to 100% of the polynucleotide. In addition, the polycationiccomplexes fuse with cell membranes, resulting in an intracellulardelivery of polynucleotide that bypasses the degradative enzymes of thelysosomal compartment. PCT publication WO 94/27435 describescompositions for genetic immunization including cationic lipids andpolynucleotides. Agents that assist in the cellular uptake of nucleicacid, such as calcium ions, viral proteins and other transfectionfacilitating agents, may be included.

Polynucleotide immunogenic preparations may also be formulated asmicrocapsules, including biodegradable time-release particles. U.S. Pat.No. 5,151,264 describes a particulate carrier ofphospholipid/glycolipid/polysaccharide nature that has been termed BioVecteurs Supra Moleculaires (BVSM).

According to the present invention, methods of this invention includeadministering an effective amount of a composition of the presentinvention as described above to the subject. The effective amount of thecomposition, the use of which is in the scope of present invention, willvary somewhat from subject to subject, and will depend upon factors suchas the age and condition of the subject and the route of delivery. Suchdosages can be determined in accordance with routine pharmacologicalprocedures known to those skilled in the art. For example, the activeagents of the present invention can be administered to the subject in anamount ranging from a lower limit from about 0.01, 0.05, 0.10, 0.50,1.0, 5.0, or 10% to an upper limit ranging from about 10, 20, 30, 40,50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% by weight of thecomposition. In some embodiments, the active agents include from about0.05 to about 95% by weight of the composition. In other embodiments,the active agents include from about 0.05 to about 60% by weight of thecomposition. In still other embodiments, the active agents include fromabout 0.05 to about 10% by weight of the composition.

In particular embodiments of the present invention, the compositiondescribed herein is immunogenic. That is, the administration of theactive agents can be carried out therapeutically (i.e., as a rescuetreatment) or prophylactically. For example, in some embodiments, toprotect against an autoimmune disease, subjects may be vaccinated inanticipation of antigen exposure, as neonates or adolescents. Adultsthat have not previously been exposed to the disease may also bevaccinated. The immunogenic composition of the present invention can begiven as a single dose schedule or in a multiple dose schedule. Amultiple dose schedule is one in which a primary course ofadministration may consist of about 1 to 10 separate doses, followed byother doses (i.e., booster doses) given at subsequent time intervals tomaintain and/or reinforce the immune response, for example, at about 1to 4 months for a second dose, and if needed, a subsequent dose(s) afteranother several months. The dosage regimen will also, at least in part,be determined by the need of the individual and be dependent upon thejudgment of the medical or veterinary practitioner.

Embodiments of the present further provide kits comprising one or morecontainers having pharmaceutical dosage units including an effectiveamount of a fusion protein or portion thereof, wherein the container ispackaged with optional instructions for the use thereof.

As described in further detail below, the present invention finds use inboth veterinary and medical applications. Suitable subjects includeavians, mammals and fish, with mammals being preferred. The term “avian”as used herein includes, but is not limited to, chickens, ducks, geese,quail, turkeys and pheasants. The term “mammal” as used herein includes,but is not limited to, primates (e.g., simians and humans), bovines,ovines, caprines, porcines, equines, felines, canines, lagomorphs,rodents (e.g., rats and mice), etc. Human subjects include fetal,neonatal, infant, juvenile and adult subjects.

4. Methods of Use

Embodiments of the present invention provide methods of regulating animmunological disorder including administering an effective amount of afusion protein including an autoimmune antigen, an allergen antigen oran alloantigen or a portion thereof and an anti-inflammatory cytokine orportion thereof. In some embodiments, the method includes administeringat least two fusion proteins that include an anti-inflammatory cytokineor portion thereof, wherein at least one fusion protein includes ananti-inflammatory cytokine or portion thereof that is different fromthat of at least one other fusion protein.

According to embodiments of the present invention, the immunologicaldisorder includes autoimmune diseases, allergic or hypersensitivitydiseases, transplant rejection, and tissue disorders.

Autoimmune diseases include, but are not limited to, those affecting thefollowing organ systems. Nervous system: Acute disseminatedencephalomyelitis (demyelinating inflammation following vaccination orinfection); Myasthenia Gravis (anti-AchR antibodies, blockade ofneuromuscular junction); Multiple sclerosis (inflammation of CNSmyelin); Acute inflammatory demyelinating polyneuropathy/Guillain-Barresyndrome (inflammation of peripheral myelin); Endocrine system:Hashimoto's Thyroiditis (anti-thyroid antibodies, hypothyroidism);Grave's Disease (auto-antibodies stimulate TSH receptors on thyroidfollicular cells, hyperthyroidism); Insulin-Dependent Diabetes Mellitus(i.e. juvenile diabetes, inflammation and deletion of β islet cells);Autoimmune adrenal insufficiency (e.g. Addison's disease, inflammationcoupled with progressive scarring and atrophy of adrenal glands);Autoimmune oophoritis (inflammation of ovaries, infertility); Autoimmuneorchitis (inflammation of testis); Hematopoietic system: Autoimmunehemolytic anemia (anti-erythrocyte antibodies); Paroxysmal coldhemoglobinuria (mediated by IgM cold agglutinins against erythrocytes);Idiopathic thrombocytopenic purpura (anti-platelet antibodies,bleeding); Autoimmune neutropenia (antibodies against neutrophils causedegranulation, neutrophil depletion, and vasculitis); Pernicious anemia(progressive destruction of gastric fundic gland, loss of intrinsicfactor, and malabsorption of vitamin B₁₂); Autoimmune coagulopathy(circulating anti-coagulants, anti-phospholipid antibody syndrome,neutralizes phospholipids necessary for clotting activity);Gastrointestinal Tract: Primary biliary cirrhosis (intrahepatic bileduct and portal inflammation leading to fibrosis and cirrhosis);Inflammatory bowel disease (Crohn's disease, ulcerative colitis);Kidney: Glomerulonephritis (antibody against glomerular basementmembrane); Immune complex glomerular nephritis (accumulation ofdeposited immune complexes in basement membrane); Skin: Pemphigusvulgaris (loss of adhesion between epidermal cells, blistering, antibodyagainst stratified squamous epithelium); Systemic autoimmune disease:Systemic Lupus Erythematosus (arthralgias, rash, nephritis, anti-nuclearantibodies); Rheumatoid Arthritis (inflammatory polyarticular arthritis,rheumatoid factor); Sjogren's syndrome (inflammation of lacrymal andparotid glands with arthritis); Polymyositis (inflammation of skeletalmuscle); Dermatomyositis (inflammation of skin and skeletal muscle);Scleroderma (progressive systemic sclerosis, sclerosis of skin andinternal organs); and Cardiac and vascular diseases: Autoimmunemyocarditis (inflammation of cardiac muscle); Immune complex-mediatedvasculitis (passive deposition of immune complexes in vessel wallsfollowed by C-mediated lysis and inflammation); Polyarteritis nodosa(type of necrotizing vasculitis that follows certain types ofinfections). In some embodiments of the present invention, theautoimmune disease is an autoimmune disease affecting the nervoussystem, endocrine system, hematopoietic system, gastrointestinal tract,renal system, cardiac system, vascular system, musculoskeletal system ora combination thereof. In some embodiments, the autoimmune disease is asystemic autoimmune disease. In particular embodiments, the autoimmunedisease is multiple sclerosis.

Allergic or hypersensitivity diseases include, but are not limited to,allergic rhinitis, asthma, atopic dermatitis, allergicgastroenteropathy, contact dermatitis, drug allergy or a combinationthereof. In particular embodiments, the present invention providesactive agents, compositions and methods to induce antigen-specificimmunological tolerance to allergens responsible for the allergicdiseases described herein.

Transplant rejection and tissue disorders include, but are not limitedto, those affecting the kidney, liver, pancreas, heart, lung, bone, skinand combinations thereof. In particular embodiments, the presentinvention provides compositions and methods to induce antigen-specificimmunological tolerance to allogeneic and xenogeneic transplantationantigens that may contribute to the rejection of tissue transplants, andthus, facilitate acceptance of kidney transplants, liver transplants,pancreas transplants, skin grafts, heart transplants, and heart-lungtransplant. The active agents and methods may also alleviatecomplications of bone marrow transplantation (i.e., graft versus hostdisease).

It is contemplated that diseases and/or disorders treated by the methodsof this invention can include any disease or disorder that can betreated by mounting an effective tolerogenic response to a fusionprotein of the invention. Accordingly, embodiments of the presentinvention provide methods of modulating an immune response includingadministering a fusion protein in an amount sufficient to elicit atolerogenic response. In some embodiments, the immune response isantigen-specific. In some embodiments, the administering step is carriedout in vivo or ex vivo. In still other embodiments, the tolerogenicresponse is an active tolerance mechanism. In particular embodiments,the tolerogenic response is a sustained tolerogenic response.

It is also contemplated that the compositions of this invention can beused as a vaccine or prophylactic composition and employed in methods ofpreventing a disease or disorder in a subject, comprising administeringto the subject an effective amount of the active agent of thisinvention. The vaccine can be administered to a subject who isidentified to be at risk of contracting a particular disease ordeveloping a particular disorder and in whom the ability to elicit animmune response to an antigen may be impaired. Identification of asubject at risk can include, for example, evaluation of such factors asfamily history, genetic predisposition, age, environmental exposure,occupation, lifestyle and the like, as are well known in the art.

The effective dosage of any specific active agent will vary somewhatfrom composition to composition, patient to patient, and will dependupon the condition of the patient and the route of delivery. As ageneral proposition, a dosage from about 0.1 to about 50 mg/kg will havetherapeutic efficacy, with still higher dosages potentially beingemployed for oral administration, wherein aerosol administration isusually lower than oral or intravenous administration. Toxicity concernsat the higher level may restrict intravenous dosages to a lower levelsuch as up to about 10 mg/kg, all weights being calculated based uponthe weight of the active base, including the cases where a salt isemployed. Typically a dosage from about 0.5 mg/kg to about 5 mg/kg willbe employed for intravenous or intramuscular administration. A dosagefrom about 10 mg/kg to about 50 mg/kg may be employed for oraladministration.

In particular embodiments, administration to a subject such as a human,a dosage of from about 0.01, 0.1, or 1 mg/kg up to 50, 100, or 150 mg/kgor more for each active agent can be employed. Depending on thesolubility of the particular formulation of active agents administered,the daily dose can be divided among one or several unit doseadministrations.

Having now described the invention, the same will be illustrated withreference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

EXAMPLES Example 1 Methods of Making Exemplary Fusion Proteins

A. Recombinant Protein Design

The structural design of the fusion proteins are shown in Table 2. Astrategy of overlap extension PCR was used to construct genes encoding agiven cytokine fused by a cleavable linker (an enterokinase domain) withthe dominant encephalitogenic sequence of guinea pig myelin basicprotein (GPMBP) and a C-terminal 6-histidine sequence. For example,IL1RA/NAg4 consisted of the native IL1RA signal sequence (ss), the fulllength IL1-RA cytokine, a GDDDDKG (SEQ ID NO:1) enterokinase (EK)domain, the encephalitogenic peptide of GPMBP (PQKSQRSQDENPVVH, SEQ IDNO:2), and a 6-his C-terminal tag. The IL2.7, IL4.4, IL10.6, and IL13.6constructs were based on a parallel design. The encephalitogenic peptidewas the 73-87 sequence of GPMBP (GP73-87). The numbering system for thispeptide of GPMBP was based on accession P25188. The upstream anddownstream primers used for amplification of the entire cytokine/NAgfusion gene contained 5′ Spe I and Kpn I restriction endonuclease sites,respectively.

Several other fusion proteins were also used as controls (Table 2).Baculovirus expression systems for rat IL-2 and IL-4 were describedpreviously (Norris et al. (2001) Cell Immunol 211, 51-60; Mannie et al.(2003) Immunol Cell Biol 81, 8-19). These proteins consisted of thenative rat cytokine without N- or C-terminal modifications. The IL2Ekdeland IL4Ekdel constructs were generated via a mutagenesis reaction thatdeleted the DNA sequence encoding the EK domain from the recombinantIL2.7 and IL4.4 pFastbac1 vectors. These proteins consisted of thenative IL-2 or IL-4 cytokine fused directly to the GP73-87 peptide andincluded the adjoining C-terminal 6his tag. The IL2-D, IL2-C, IL4-A,IL13-A, IL13-B proteins consisted of the native cytokine directly fusedto a C-terminal 6his tag. These latter proteins lacked the Ek-NAgsequence and thereby served as relevant comparisons to assess thefunction of the NAg domain in cytokine/NAg fusion proteins. Primers(Table 3) were designed based on DNA sequences for rat cytokines IL-1RA(Eisenberg et al. (1991) Proc Natl Acad Sci USA 88, 5232-5236)(accession NM_(—)022194), IL-2 (McKnight et al. (1989) Immunogenetics30, 145-147) (M22899), IL-4 (McKnight et al. (1991) Eur J Immunol 21,1187-1194) (X16058), IL-10 (Feng et al. (1993) Biochem Biophys ResCommun 192, 452-458) (NM_(—)012854), and IL-13 (Lakkis et al. (1993)Biochem Biophys Res Commun 197, 612-618) (L26913).

B. RNA Isolation, cDNA Synthesis, Overlap Extension PCR of Cytokine/NAgConstructs

Lewis rat splenocytes (3×10⁶/ml) were activated in complete RPMI with2.5 μg/ml Con A and 10 μg/ml LPS for 24 hours. Total RNA was purified byuse of the TRIzol® Reagent (Invitrogen). Rat cytokine cDNA wassynthesized in the presence of 200 nM downstream primer (3′ primers, seeTable 3), 1 μg total RNA, first strand buffer, dNTP, DTT, and eitherSuperScript II RNase H—Reverse Transcriptase or SuperScript III ReverseTranscriptase (Invitrogen) according to manufacturer's instructions in atotal volume of 20 μl. To amplify the cytokine gene, 10 μl of the RTproduct was mixed with 100 nM upstream primer (5′ primers) and PlatinumTaq DNA Polymerase High Fidelity (Invitrogen) together with PCR mix,MgSO₄, and dNTP. The downstream primer was carried over in the RTproduct at a final concentration of 100 nM. The PCR cycle was 94° C. 1′,59° C. 1′, and 68° C. 1′ for 39 cycles followed by a final 68° C.-8′extension step. The PCR products were washed 3 times with H₂0 by use ofMicrocon YM100 Centrifugal Filter Devices.

To generate the neuroantigen, 100 nM 5A01 primer and 100 nM 2F07 primerwere mixed and extended with Platinum Pfx DNA polymerase (Invitrogen) inthe presence of PCR buffer, MgSO₄, and dNTP according to manufacturer'sinstructions. The PCR cycle was 94° C. 1′, 62° C. 1′, and 68° C. 5′ for10 cycles followed by a final 68° C.-10′ extension step. The resultingDNA encoded the following peptide sequence: DDDDKGPQKSQRSQDENPVVHHHHHH(SEQ ID NO:3) and included a Kpn I endonuclease restriction site. Thisoverlap extension reaction is portrayed in FIG. 1, Panel A. The DDDDK(SEQ ID NO:4) sequence represents an enterokinase cleavage site and the15mer sequence PQKSQRSQDENPVVH (SEQ ID NO:2) represents the dominantencephalitogenic region of guinea pig myelin basic protein. To generatethe IL2NAg, IL4NAg, IL10NAg, IL13NAg cytokine/NAg fusion constructs,1/20 dilution of cytokine PCR product (2 μl) was mixed with a 1/20dilution of the NAg PCR product (2 μl), 200 nM of respective upstreamprimer, and 200 nM of the 2F07 downstream primer, and the reactants wereamplified by the Platinum Pfx DNA polymerase. The overlap extension PCRused to generate the IL2.7 (IL2NAg) construct is diagramed in FIG. 1,Panel B. The PCR cycle was 94° C. 1′, 57° C. 1′, and 68° C. 2′ for 39cycles followed by a final 68° C.-10′ extension step.

To generate the IL1RANAg fusion construct, 10 μl of the respective RTproduct was mixed with 200 nM of 3B05 upstream primer together with 100nM 5A01 and 200 nM 2F07 primer. In this reaction, the 5A02 downstreamprimer was carried over in the RT product at a final concentration of100 nM. In the latter reaction, the cytokine, the NAg, and thecytokine/NAg construct were amplified in a single PCR reaction catalyzedby Platinum High Fidelity Taq DNA Polymerase.

To generate the IL2Ekdel construct, the IL2.7 pFastbac1 plasmid was usedas template for a whole plasmid PCR in the presence of the 5H04 and 6E12primers and Platinum Pfx DNA polymerase. 5H04 was the mutagenic primerthat lacked the coding sequence for the EK domain. This primer and the6E12 primer were overlapping primers that generated linearized copies ofthe plasmid. To generate the IL4Ekdel construct, the IL4.4 pFastbac1plasmid was used as template for a whole plasmid PCR in the presence ofthe 5H05 and 6F01 primers. The 5H05 primer was mutagenic by virtue oflacking the coding sequence for the EK linker. In both of the IL2Ekdeland IL4Ekdel reactions, the reaction mix was melted at 95° C. for 5′followed by 29 cycles at 94° C. for 30″, 60° C. for 1′, 68° C. for 3′followed by a 68° C. 10′ extension step. The products were washed andconcentrated by use of Microcon YM100 Centrifugal Filter Devices, weretreated with Dpn I restriction endonuclease to deplete residual IL2.7and IL4.4 parental plasmids, and were used to transform Top10 E coli byelectroporation. Colonies bearing recombinant pFastbac1 plasmids withdeletion of the EK coding region were identified by restrictiondigestion of purified plasmids and analysis on 1.5% agarose gels.Deletion of the EK coding region in the respective plasmids was verifiedby DNA sequencing of the cytokine-NAg-6his fusion gene.

To generate the IL2woNAg (IL2-D, IL2-C), IL4woNAg (IL4-A), and IL13woNAg(IL13-A, IL13-B) constructs, purified IL2.7, IL4.4, and IL13.6 plasmidswere used as templates, and PCR reactions were run with upstream primers3B12, 2D11, or 2E01 and downstream primers 6H02, 6H04, and 6H06,respectively (Table 3). The latter three primers had a 5′-Kpn Irestriction together with the coding region for the 6his tag and the 5′sequence of the cytokine gene. The coding region for the EK-NAg sequencewas deleted from these downstream primers. These PCR products therebycontained sequences encoding the respective cytokine with a 6hisC-terminal tag. These PCR products were cut with Spe I and Kpn Irestriction endonucleases and were cloned into the pFastbac1 vector forgeneration of recombinant baculovirus.

C. Derivation of Baculovirus Expression Systems

The IL1RANAg (636 bp), IL2NAg (570 bp), IL4NAg (543 bp), IL10NAg (639bp), and IL13NAg (495 bp) PCR fusion products exhibited the sizespredicted for each fusion construct. The PCR products were purified on a1% TAE agarose gel, and DNA was extracted from gel slices by use ofAmicon Ultrafree DA Units. PCR products were washed 3 times with H₂O byuse of Microcon YM100 Centrifugal Filter Devices. These PCR productswere digested with SpeI and KpnI restriction endonucleases (Invitrogen)for 4 hrs at 37° C. according to manufacturer's instructions. Digestswere heat-inactivated at 70° C. for 10′ and extensively washed by use ofMicrocon YM100 Centrifugal Filter Devices. Likewise, purified pFastBac1plasmid (Invitrogen) was digested with Spe I and Kpn I, was purified ona 1% TAE agarose gel, and then extensively washed with Microcon YM100devices.

To ligate insert DNA into the vector, 90 fmoles of cytokine/NAg DNA weremixed with 30 fmoles of vector plasmid in the presence of T4 DNA ligasefor 1 hr at room temperature. Ligation mixes were then used to transformTop10 E. coli by electroporation. Colonies exhibiting growth in thepresence of 100 μg/ml ampicillin were grown in minicultures. Plasmidswere purified, digested with Spe I and Kpn I, and assessed for theappropriate sized inserts on 1.5% agarose gels. Plasmids bearing theappropriate sized inserts were subject to forward and reverse DNAsequencing to verify the predicted sequence of the fusion gene.

Purified recombinant pFastBac1 plasmids were used to transformelectrocompetent DH10BAC E. coli to facilitate site-specifictransposition of the respective expression cassettes into a baculovirusshuttle vector according to modified protocols of the Bac-to-BacBaculovirus Expression System (Invitrogen). White/blue selectionfacilitated identification of recombinant DH10BAC colonies. Bacmid DNAcontaining the recombinant baculovirus shuttle vector was purified andused to transfect Sf9 insect cells according to the manufacturer'sprotocol. Establishment of recombinant baculovirus was confirmed by theappearance of an infected morphology of Sf9 insect cells in 3-7 daycultures. The presence of recombinant baculovirus were verified by PCRanalysis for the presence of the respective cytokine/NAg DNA.Recombinant baculovirus were also verified by the presence of cytokineand NAg activity in supernatants of infected Sf9 cells.

D. Confirmation of Recombinant Baculovirus

A standard bioassay was used to analyze supernatants of P1 (passage #1)baculovirus stocks for the presence of secreted fusion proteins. Thebioassay measured whether titrations of the P1 supernatants hadantigenic activity. The presence of the NAg domain in the supernatantswas measured by the antigen-dependent proliferation of the MBP-specificRsL.11 T cell clone. That is, supernatants from infected Sf9 cultureswere titrated in complete RPMI medium followed by the addition of RsL.11T cells (25,000/well) and irradiated splenic antigen presenting cells(500,000/well). After 2 days of culture, cultures were pulsed with 1 μCiof [³H]thymidine and were harvested after an additional 24 hr ofculture. Antigen-specific proliferation was assessed by [³H]thymidineincorporation in triplicate or quadruplicate cultures.

E. Purification of Recombinant Baculovirus

Titrations of baculovirus infected Sf9 supernatant from 10⁻⁴ to 10⁻⁹were added to Sf9 cells (10⁴ cells/well in 96 well flat bottomed plates)in Sf900 II SFM medium. Each titration was added to approximately 24separate wells. In 7-10 days, wells were visually inspected for thepresence of an infected cellular morphology, and cultures showinglimiting dilution growth of baculovirus was identified as those thatlikely originated from a single baculovirus particle. Baculovirussupernatants (10 μl) were transferred to replicate plates containingRsL.11 T cells (25,000/well) and irradiated splenocytes (500,000/well).T cell proliferation was measured by [³H]thymidine incorporation.Titrations exhibiting limiting dilution growth of baculovirus and highlevels of antigenic activity were harvested and used at a titration of1/2000 to infect new Sf9 cultures to establish purified baculovirusstocks. These baculovirus stocks were then used to support proteinexpression experiments.

F. Purification of Fusion Proteins

To purify fusion proteins, baculovirus supernatants were concentrated onYM10 ultrafiltration membranes and then subjected to two consecutiveaffinity chromatography steps. The first affinity chromatography stepwas described in (Blank et al. (2002) Protein Expr Purif 24, 313-322)and was based on a plasmid generously provided by Drs. Peter Lindner andAndreas Pluckthun. This plasmid encoded a single chain Fv anti-6hisantibody fused to two tandem chitin-binding domains (scFv-CBD2). Afterexpression in E. coli, bacterial lysates were passed through 1 ml ofchitin resin column by gravity flow. This step enabled purification ofthe scFv-CBD2 protein via binding of the tandem chitin binding domainsto the chitin bead resin. Thus, the anti-6his single chain antibody wasimmobilized onto chitin columns and was used to purify recombinantproteins bearing C-terminal 6-histidine tags from concentratedbaculovirus supernatants. These affinity chromatography columns werestored in TBST buffer (50 mM TrisHCl, 500 mM NaCl, 0.1 mM EDTA, 1%Triton X-100, 0.1% Na azide, pH 8.0). After equilibration in MBS buffer(20 mM MES, 500 mM NaCl, 0.1 mM EDTA, pH 6.5), concentratedbaculovirus-infected Sf9 supernatants were passed through the column totrap the cytokine/NAg fusion protein. Interactions of the scFv-CBD2 with6his-tagged proteins were stable in weak acids (pH5.5) but weredisrupted in basic solutions (pH˜10), and cytokine/NAg proteins wereeluted in CAPS buffer (50 mM CAPS, 500 mM NaCl, 0.1 mM EDTA, pH 10.0).The second affinity chromatography step involved direct loading of thechitin column eluate onto nickel columns (Qiagen) followed by extensivewashing of the Ni resin (50 mM NaH₂PO₄, 500 mM NaCl, 10 mM imidazole, pH8.0) and elution with 250 mM imidazole (pH 8.0) or by acid elution (pH4.0). Protein quantity was assessed by the BCA protein assay (Pierce)and purity was assessed by SDS-PAGE.

Example 2 Biological Activity

a. Animals and Reagents

Lewis rats were bred and maintained at East Carolina University Schoolof Medicine. Care and use of experimental animals was performed inaccordance with institutional guidelines. GPMBP was purified from guineapig spinal cords (Rockland). OX6 anti-I-A (RT1B) IgG1, OX17 anti-1-E(RT1D) IgG1 (McMaster and Williams. (1979) Immunological Reviews 47,117-137), OX39 anti-IL2Ralpha IgG1 (Paterson et al. (1987) Mol Immunol24, 1281-1290) were concentrated by ultrafiltration of B cell hybridomasupernatants through Amicon spiral wound membranes (100 kDa exclusion).Hybridomas were obtained from the European Collection of Cell Cultures.FITC-conjugated goat anti-mouse IgG1 reagents were purchased fromSouthern Biotechnology (Birmingham, Ala.). The domain structures ofcytokine/NAg fusion proteins used in this study are shown in Table 2.The synthetic peptide GP69-88 (YGSLPQKSQRSQDENPVVHF) was obtained fromQuality Controlled Biologicals, Inc. (Hopkinton, Mass.).

B. Cell Lines and Culture Conditions

The RsL.11 MBP-specific clone was derived from Lewis rats sensitizedwith rat MBP in CFA. The R1-trans T cell clone was a transformed, IL-2dependent line that constitutively expressed MHCII, B7.1, and B7.2(Patel et al. (1999) J Immunol 163, 5201-5210; Mannie and Norris (2001)Cell Immunol 212, 51-62). The GP2 line and the derivative GP2.3H3.16clone was derived from Lewis rats sensitized with GPMBP (Mannie et al.(1998) Cell Immunol 186, 83-93; Walker et al. (1999) J Leukoc Biol 66,120-126). Assays were performed in complete RPMI medium [10%heat-inactivated fetal bovine serum (Summit, Boulder, Colo.), 2 mMglutamine, 100 μg/ml streptomycin, 100 U/ml penicillin (WhittakerBioproducts, Walkersville, Md.), 50 uM 2-ME (Sigma)]. T cell lines werepropagated in complete RPMI supplemented with recombinant rat IL-2 (0.4%v/v Sf9 supernatant). Con A (Sigma-Aldrich, St. Louis, Mo.) was used ata final concentration of 2.5 μg/ml.

C. Proliferative Assays

Cultures were pulsed with 1 μCi of [³H]thymidine (6.7 Ci/mmol, NewEngland Nuclear) after 48 hours of a 3-day assay. Cultures wereharvested onto filters by use of a Tomtec Mach III harvester.[³H]thymidine incorporation into DNA was measured by use of a Wallac1450 Microbeta Plus liquid scintillation counter. Error bars representstandard deviations of triplicate or quadruplet sets of wells.

D. Measurement of IL-2 and Nitric Oxide

To measure IL-2 bioactivity, CTLL cells (10⁴ cells/100 μl completeRPMI/well) were cultured with designated supernatants for 48 hours, and10 μl of a MTS/PMS solution [2.9 mg/ml MTS (Promega) and 0.1 mg/ml PMS(Sigma)] were added to each well. Plates were read the next day at 492nm on an Anthos ELISA Reader (ACCSales, Chapel Hill, N.C.).Antigen-stimulated IL-2 production was calculated as the mean OD valuesfrom experimental cultures minus the mean OD values from controlunstimulated cultures. The production of nitric oxide was measured byformation of the stable decomposition product nitrite in cell-freesupernatants (50 μl) after mixing with an equal volume of the Griessreagent (Ding et al (1988) J Immunol 141, 2407-2412).

E. Flow Cytometric Analysis

T cells were incubated with a 1/20 titration of a concentratedsupernatant containing OX6, OX17, or OX39 IgG1 mAb for 45 minutes at 4°C. The cells were washed two times and were incubated for 45 minuteswith FITC-conjugated goat anti-mouse IgG1 reagents. Dead cells wereexcluded from analysis by forward versus side scatter profiles. Datawere acquired with a Becton Dickinson FACScan flow cytometer and wereanalyzed with the CELLQuest software program.

F. Tolerance Induction

To determine whether cytokine/NAg proteins prevent active induction ofexperimental autoimmune encephalomyelitis (EAE) in Lewis rats, rats weregiven a total of three subcutaneous injections of a given cytokine/NAgprotein. A dose of 0.5 to 1 nmole of cytokine/NAg was delivered duringeach injection at 1-2 week intervals as designated. The cytokine/NAgproteins were either solubilized in saline or were emulsified in alum.At least 7 days after the last injection, rats were challenged with NAgin Complete Freund's adjuvants (CFA) (day 0) to induce EAE.

G. Induction and Clinical Assessment of EAE

EAE was induced in Lewis rats by injection of an emulsion containing 25or 50 μg GPMBP in CFA (200 μg Mycobacterium tuberculosis). In designatedexperiments, rats were challenged with an emulsion containing 50 μg ofthe DHFR-NAg fusion protein in CFA. DHFR-NAg was comprised of the mousedihydrofolate reductase as the N-terminal domain and theencephalitogenic GP69-87 peptide of GPMBP as the C-terminal domain. Theemulsion (total volume of 0.1 ml per rat) was injected in two 0.05 mlvolumes on either side of the base of the tail. The following scale wasused to assign intensity of EAE; paralysis in the distal tail, 0.25;limp tail, 0.5; ataxia, 1.0; hind leg paresis, 2.0; full hind legparalysis, 3.0. The cumulative score for each rat consisted of the sumof daily scores for each animal. The mean cumulative score for a groupwas calculated by averaging the cumulative scores for all rats within agroup. The mean maximal intensity scores were assigned to each groupbased on the average maximum score among afflicted rats within a group.

H. Lewis Rat T Cells

The RsL.11 T cell and the R1-trans clone were described previously(Mannie and Norris. (2001) Cell Immunol 212, 51-62). T cells wereassayed in complete RPMI medium and were maintained in same mediumsupplemented with recombinant rat IL-2. The complete RPMI 1640 mediumconsisted of 10% heat-inactivated fetal bovine serum, 2 mM glutamine,100 μg/ml streptomycin, 100 U/ml penicillin (Whittaker Bioproducts,Walkersville, Md.), and 50 μM 2-ME (Sigma). Rat IL-2 was obtained from arecombinant baculovirus expression system (Mannie et al. (2003) ImmunolCell Biol 81, 8-19).

I. In Vitro Proliferation

Responder T cells (2.5×10⁴/well) were cultured with irradiatedsplenocytes (5×10⁵/well) and with designated concentrations of antigen.After 2 days of culture, T cells were pulsed with 1 μCi of [³H]thymidine[6.7 Ci/mmol, Perkin-Elmer (Boston, Mass.)]. After another 1 day ofculture, T cells were harvested onto filters to measure [3H]thymidineincorporation by scintillation counting. Error bars portray standarddeviations.

J. Cytokine-Specific Activity of Cytokine/NAg Fusion Proteins

Recombinant baculovirus were used to infect Sf9 insect cells, andsupernatants from infected cultures were tested for expression of therespective cytokine activities. These experiments (FIGS. 2-7) revealedthat the cytokine domain of the respective cytokine/NAg fusion proteinshad intact cytokine-specific biological activity.

Supernatants containing IL1RA/NAg4 blocked IL-1 activity (FIG. 2). Inaccordance with a widely-used bioassay for IL-1RA (Arend et al. (1989) JImmunol 143, 1851-1858), IL-1 augmented the proliferation ofmitogen-stimulated thymocytes, and the IL1 RA/NAg4 fusion proteinblocked the ability of IL-1 to augment mitogen-stimulated proliferation.The IL1RA/NAg4 baculovirus system inhibited over 90% of IL-1 augmentedproliferation. At all titrations tested, the IL1RA/NAg baculovirussupernatant did not significantly inhibit Con A-stimulated proliferationin the absence of recombinant human IL-1β. Thymocyte proliferation inresponse to Con A or the combination of Con A & IL-1 is shown in FIG. 2,Panel A. The same data are portrayed in FIG. 2, Panel B as percentinhibition of IL-1 stimulated growth versus percent inhibition of ConA-stimulated growth. These data indicated that IL1 RA/NAg4 blocked theaction of IL-1 but did not interfere with Con A-stimulated mitogenesisper se. These findings were consistent with the predicted bioactivitiesof IL1 RA, and the specificity of the inhibitory effect discounted anonspecific inhibitory mechanism.

Four different IL-2 fusion proteins exhibited equivalent IL-2 activityin a CTLL bioassay (Gillis et al. (1978) J Immunol 120, 2027-2032) (FIG.3, Panel A) when the respective baculovirus supernatants were titratedfrom 10% (10⁻¹) to approximately 0.00001% (10⁻⁷). The exponent of thetitration was plotted on the x-axis. The C-terminal addition ofEk-GP-6his (IL2.7), GP-6his (IL2Ekdel), or 6his (IL2-D) domains did notappear to affect IL-2 bioactivity or the efficiency of the baculovirusexpression system. A normative Ala² was inserted into the signalsequence after the N-terminal Met in IL2.7, IL2Ekdel, IL2-D, & IL2-C toprovide an optimal Kozak translation initiation sequence (Kozak (1997)Embo J 16, 2482-2492). This structural alteration however appeared to bewithout consequence (FIG. 3, Panel A). A single mutation (Y¹⁴⁶ for C¹⁴⁶)in the IL2-C protein however was associated with an approximate 1,000fold reduction in activity (FIG. 3, Panel B). Because supernatantsrather than purified protein were assessed, the reduction in activitymay be associated with decreased expression, stability, or activity ofthe mutant protein. By homology to human IL-2 (Wang et al. (1984)Science 224, 1431-1433), rat IL-2 likely has a single disulfide bondbetween C⁷⁸ and C¹²⁶ and a single unpaired C¹⁴⁶. The substitution ofY¹⁴⁶ for C¹⁴⁶ was a chance mutation detected during DNA sequencing ofrecombinant pFastbac1 plasmids. In prokaryotic expression systems forhuman or murine IL-2, this unpaired Cys was deliberately substitutedwith Ser or Ala to prevent inappropriate disulfide bond formation, andthese substitutions did not compromise biological activity (Wang et al.(1984) Science 224, 1431-1433; Liang et al. (1986) J Biol Chem 261,334-337). However, the Y¹⁴⁶ for C¹⁴⁶ mutation in rat IL-2 was not welltolerated.

IL-4 is a known T cell growth factor (Yokota et al. (1986) Proc NatlAcad Sci USA 83, 5894-5898; Mitchell et al. (1989) J Immunol 142,1548-1557). Accordingly, the IL4.4 cytokine/NAg fusion protein, likeIL2.7, had mitogenic activity in thymocyte cultures that wereco-stimulated with PHA and IL-113 (FIG. 4, Panel A). Baculovirussupernatants containing these proteins exhibited mitogenic activity attitrations as low as 0.02% (10^(−3.7)). The mitogenic activity wasattributed to IL-2 and IL-4, because fusion proteins incorporating othercytokines [IL1 RA (not shown), IL-10, or IL-13] were without activity.IL-4 activity was distinguished from IL-2 by assays in which thymocyteswere co-stimulated with IL-2 and PMA (FIG. 4, Panel B). IL2.7 did notstimulate additional proliferation in this assay because IL-2 waspresent in saturating concentrations in all wells. In this assay, IL4.4and a sister clone IL4.8 stimulated thymocyte proliferation whereas theIL2.7, IL10.6, and IL13.6 fusion proteins had no activity.

Consistent with the known activities of IL-4 and IL-13 (Doherty et al.(1993) J Immunol 151, 7151-7160; Doyle et al. (1994) Eur J Immunol 24,1441-1445; Bogdan et al. (1997) J Immunol 159, 4506-4513), the IL4.4 andIL13.6 fusion proteins (native ss-cytokine-EK-GP-6his) also inhibitednitric oxide production by γ IFN-stimulated macrophages. When addedtogether into the same culture, the IL4.4 and IL13.6 fusion proteinsresulted in an approximate nonadditive inhibition of nitric oxideproduction (FIG. 5, Panels A and B). This observation is consistent withthe existence of a shared signal transduction pathway involving a commonIL-4R alpha chain in the respective receptor signaling complexes(Aversa, G et al. (1993) J Exp Med 178, 2213-2218; Mentink-Kane and Wynn(2004) Immunol Rev 202, 191-202; Arima et al. (2005) J Biol Chem 280,24915-24922). IL4.4 and IL13.6 were the only fusion proteins capable ofinhibiting γIFN-induced nitric oxide production whereas other fusionproteins such as IL2.6, IL10.6, and IL1RA/NAg4 lacked inhibitoryactivity (FIG. 5, Panel C and data not shown). IL2.6 and IL2.7 proteinswere derived from sister clones and had identical protein sequence.These data verify IL-4 and IL-13 as cytokines that modulate macrophageactivity.

Unlike IL4.4, IL13.6 had no activity on T cells. Both IL4.4 and IL4(without NAg) profoundly inhibited the IL-2 dependent growth of theMBP-specific RsL.11 clone and the conalbumin-specific Conal.8D9 clone(FIG. 6, Panels A and B) whereas IL4.4, IL4, and IL4-A elicited T cellgrowth in thymocyte cultures costimulated with IL-2 and PMA (FIG. 6,Panel C). These data indicated that IL-4 receptors exist on both IL-2dependent lines of memory rat Th1 T cells and on naïve thymocyes andmediate inhibitory or stimulatory action according to the differentiatedfunction of either subset. In contrast, the IL-13 fusion proteins(IL13.6, IL13-A, nor IL13-B) did not modulate the proliferation of theIL-2 dependent T cell lines (RsL.11 or Conal.8D9) and did not affect thegrowth of thymocytes even though IL13-based fusion proteins inhibitednitric oxide production by macrophages (FIG. 6, Panel D). These data areconsistent with previous reports indicating that IL4 receptors but notIL-13 receptors are present on T cells, whereas both IL-4 and IL-13receptors exist on macrophages (Zurawski and de Vries (1994) ImmunolToday 15, 19-26).

In accordance with the ability of IL-10 to augment IL-2 dependent T cellproliferation (MacNeil et al. (1990) J Immunol 145, 4167-4173),Baculovirus supernatants containing the IL10.6 fusion protein (nativess-IL10-EK-GP-6his) augmented the IL-2 dependent growth of several rat Tcell lines, including that of the GP.3H3.16 clone, the RsL.11 clone, andthe GP2 line (data not shown). IL10.6 augmented IL-2 dependent growtheven at high concentrations (1 nM) of IL-2 but had no stimulatory effecton T cell growth in the absence of IL-2 (FIG. 7, Panel A). In RsL.11 Tcell cultures supplemented with IL-2, IL10.6 also increased cell size asmeasured by forward scatter (FIG. 7, Panel B, left) and augmentedexpression of the IL-2Rα chain (OX39 marker) whereas other cytokine/NAgfusion proteins had no activity in this assay (FIG. 7, Panel B, right).These data are consistent with the known ability of IL-10 to augmentIL-2 dependent T cell growth and facilitate expression of IL-2 receptorson T cells.

Together, these data (FIGS. 2-7) indicate that cytokine domains in theIL1RANAg, IL2NAg, IL4NAg, IL10NAg, and IL13NAg fusion proteins arebiologically active. The baculovirus expression systems resulted in theefficient secretion of these fusion proteins into the supernatant.

K. Antigen-Specific Activity of Cytokine/NAg Fusion Proteins

The baculovirus expression system coupled with a two-step affinitychromatography procedure resulted in yields of approximately 0.5-2.0 mgof purified protein per 400 ml starting volume of culture. Proteinfractions were qualitatively assessed on 12% SDS-PAGE gels, weretypically 90% pure, and exhibited a size range and banding patternconsistent with the predicted MW and potential number of N-linkedglycosylation sites (FIG. 8). Recombinant proteins secreted from Sf9insect cells are known to contain high mannose structures (Altmann etal. (1999) Glycoconj J 16, 109-123; Chang et al. (2003) J Biotechnol102, 61-71). Over-expression of recombinant protein may overwhelm theN-glycosylation machinery and lead to incomplete addition of highmannose sugars. Several of the cytokine/NAg proteins; IL4.4 and IL13.6in particular, showed MW patterns consistent with incomplete N-linkedglycosylation.

The cytokine/NAg proteins exhibited antigenic activity when tested inthe presence of the RsL.11 T cell clone and irradiated splenic APC (FIG.9, Panel A). The IL4Ekdel (IL4/GP73-87/6his) protein was at least 1000times more potent as an antigen than GPMBP. Likewise, IL4.4 had anenhanced antigenic potency that was similar to IL4Ekdel (data notshown). Fusion proteins IL2Ekdel, IL10.6, and IL13.6 had antigenicpotency that was commensurate to that of GPMBP whereas IL1RA/NAg4 hadpotency that was typically about 10-fold less than GPMBP. Thestimulatory activity of these cytokine/NAg fusion proteins in this assaywas entirely dependent upon MHC class II glycoproteins because ananti-MHCII I-A IgG1 mAb (OX6) abrogated the proliferative response. Incontrast, the OX17 mAb anti-MHCII I-E IgG1 mAb had no inhibitory action(data not shown). Therefore, this assay measured antigenic rather thancytokine dependent responses.

Even though IL-2 is known to drive the proliferation of RsL.11 T cells,IL-2 was only effective when RsL.11 T cells were activated and expressedhigh levels of IL-2 receptor. When RsL.11 T cells were quiescent, asthey were at the outset of this assay (FIG. 9, Panel A), these T cellsdid not respond to IL-2. Rather, engagement of the T cell antigenreceptor was required to enable IL-2 responsiveness. Overall, these data(FIG. 9) indicate that these cytokine/NAg fusion proteins, includingIL2NAg, are efficiently processed by splenic APC for presentation of theNAg on MHCII glycoproteins.

These fusion proteins were also tested in the presence of draining lymphnode cells (LNC) obtained from Lewis rats sensitized with DHFR/NAg inCFA. The DHFR/NAg protein was a recombinant protein bearingdihydrofolate reductase as a N-terminal domain fused to the C-terminalGP69-87 peptide of GPMBP. The IL1 RA/NAg4, IL13.6, and GPMBP proteinsstimulated approximately 30,000 cpm of [³H]thymidine, and the OX6 mAbblocked this response (background of ˜5,000 cpm) (FIG. 9, Panel B). Inthe case of IL2Ekdel, IL4Ekdel, and IL10.6, the fusion proteinsstimulated mixed responses that were in part due to the cytokine domainand in part due to antigenic stimulation of NAg-specific T cells. Forexample, 100 nM IL2Ekdel stimulated approximately 156,000 cpm and100,000 cpm of [3H]thymidine incorporation in the absence or presence ofOX6 respectively. The same concentration of IL4Ekdel stimulatedapproximately 71,000 cpm and 29,000 cpm of [³H]thymidine incorporationin the absence or presence of OX6 respectively.

These fusion proteins were also tested in an MHCII-dependent assay of Tcell-mediated killing. In these experiments, blastogenic MHCII⁺ T cellsfrom the R1-trans T cell clone were APC. Previous studies (Patel et al.(1999) J Immunol 163, 5201-5210; Mannie and Norris (2001) Cell Immunol212, 51-62; Patel et al. (2001) Cell Immunol 214, 21-34) showed thatpresentation of NAg (or conalbumin) by R1-trans T cells to irradiatedMBP-specific (or conalbumin-specific) responders resulted inantigen-specific killing of R1-trans T cells by a MHCII-restrictedmechanism. In this assay, R1-trans T cells grew rapidly in the presenceof IL-2 unless these T-APC were killed by irradiated RsL.11 responders.As shown in FIG. 10, Panel A, 1 μM GPMBP completely inhibited the IL-2dependent proliferation of R1-trans T cells, and the anti-I-A MHCII mAbOX6 abrogated this inhibition by preventing antigen recognition. In thisassay of MHCII-dependent T cell-mediated killing, the IL2Ekdel proteinwas more potent than GPMBP or DHFR-NAg by at least 5 orders of magnitude(FIG. 10, Panel B). Indeed, the IL2Ekdel fusion protein was fully activeat the lowest concentration tested—100 fM. The IL4Ekdel protein wasapproximately 1000 times more potent than GPMBP. These data indicatethat the cytokine domains of the IL2Ekdel and IL4Ekdel fusion proteinsmay potentiate antigen presentation based on high affinity interactionswith the respective cytokine receptors on the surfaces of APC.

L. Cytokine/NAg Protects Against the Subsequent Active Induction of EAE:

Subcutaneous injection of the cytokine/NAg fusion proteins in saline orin alum did not cause EAE and did not cause adverse reactions or signsof inflammation at the injection site. The following scale was used toassign intensity of EAE; paralysis in the distal tail, 0.25; limp tail,0.5; ataxia, 1.0; hind leg paresis, 2.0; full hind leg paralysis, 3.0.The cumulative score for each rat consisted of the sum of daily scoresfor each animal. The mean cumulative score for a group was calculated byaveraging the cumulative scores for all rats within a group. Meanmaximal intensity was calculated for each group based on the averagemaximum score among afflicted rats within a group. The mean severity ofEAE (y-axis of graphs) was the average score of all rats in a group on agiven day.

Three fusion proteins (IFNβNAg, NAgIL16, IL2NAg) proved to be effectivetolerogens as measured by the development of resistance to thesubsequent induction of EAE. IFNβNAg (referred to IFN 3.4) was one ofthese tolerogenic proteins. Rats were pretreated with saline or with 1nmole GP69-88 (encephalitogenic peptide YGSLPQKSQRSQDENPVVHF, SEQ IDNO:33) or 1 nmole IFNβ.4 (native ss-IFNβ-EK-NAg-6his) on days −21, −14,−7 and were challenged with 50 μg DHFR-NAg in CFA on day 0. DHFR-NAg isa fusion protein comprised of murine dihydrofolate reductase fused withthe encephalitogenic GP69-87 peptide. DHFR-NAg was derived in ourlaboratory as prokaryotic expression system for the encephalitogenicpeptide (Qiagen). DHFR-NAg has the full encephalitogenic activity ofGPMBP (not shown).

Rats pretreated with the IFNβ.4 fusion protein had less severe EAEcompared to rats pretreated with either GP68-89 or saline (Table 4).These data show marked inhibition of clinical EAE by IFNβ.4.

Subcutaneous injection of the IL2NAg or IL4NAg fusion proteins in salineor in alum did not cause EAE and did not cause adverse reactions at theinjection site. Pretreatment of rats with IL2NAg (IL2.7) in saline oralum significantly attenuated the subsequent induction of EAE (Table 5).A total of three subcutaneous injections of IL2.7 (0.5 or 1 nmole perinjection) were administered to each rat over the course of 1-2 months,and then rats were then challenged with GPMBP in CFA to elicit EAE.Pretreatment of 12.7 in saline or alum significantly reduced the meancumulative score and the mean maximal intensity of EAE. Whenadministered in saline, IL2.7 also resulted in a delayed onset ofdisease. The mechanism by which IL2.7 inhibited EAE most likely involvedtolerance induction because at least one week elapsed between the lastfusion protein injection and the encephalitogenic challenge—a delay thatallowed ample time for clearance of the fusion proteins beforechallenge.

The tolerogenic activity of the IL-2NAg fusion protein was contingentupon the covalent linkage of IL-2 and NAg (Table 6). Rats pretreatedwith IL2Ekdel had a lower mean cumulative score and a reduced meanmaximal intensity compared to rats pretreated with saline, theencephalitogenic GP69-88 synthetic peptide, purified rat IL-2, or thecombination of IL-2 and GP69-88. Rats pretreated with IL2Ekdel also hada delayed onset of EAE compared to rats pretreated with saline, GP69-88,or IL-2 alone. These findings indicated that the tolerogenic activity ofIL2NAg was antigen-specific rather than a cytokine-dependent,antigen-nonspecific activity. The tolerogenic activity of IL2NAg alsocould not be attributed to the antigen alone. Rather, the tolerogenicactivity reflected a synergy attributed to the covalent linkage of IL-2and NAg. Lastly, these data provide evidence that two independentlyderived IL2NAg fusion proteins, (IL2.7, Table 5 and IL2Ekdel, Table 6)had tolerogenic activity.

Rats challenged with DHFR-NAg in CFA often had a single relapse markedby a milder second course of EAE (FIG. 11). Pretreatment with IL2Ekdelcompletely prevented relapse in rats challenged with DHFR-NAg whereasover 50% of rats in the other four groups had spontaneous relapses. Thefinding of reduced disease severity in primary EAE and abrogation of EAEin the subsequent relapse indicated that the tolerogenic consequences ofIL2NAg treatment endured for over one month.

When delivered in either saline or alum, the IL4.4 fusion protein lackedtolerogenic activity (Table 7). Rats were given subcutaneous injectionsof IL4.4, IL2.7, or GPMBP at a dose of 1 nmole on day −42, −28, and −14,and then were challenged with 25 μg GPMBP in CFA on day 0. Again,pretreatment of rats with IL2.7 significantly decreased the meancumulative score and the mean maximal intensity of EAE and delayed theonset of EAE compared to control groups. These data indicate that IL-2is more efficient than IL-4 as a fusion partner for induction oftolerance to NAg. When cytokine/NAg fusion proteins were injected inalum rather than saline (Tables 5 & 6), the encephalitogenic challengeresulted in an accelerated onset of EAE. The ability of alum toaccelerate the course of disease was independent of the ability of IL2.7to suppress disease. Thus, pretreatment with IL2.7 in alum decreasedincidence and severity of EAE but nonetheless accelerated disease onset.

IL2NAg (IL2Ekdel) was also substantially more tolerogenic than IL4NAg(IL4Ekdel) when delivered after encephalitogenic challenge. Rats weresensitized with DHFR-NAg in CFA on day 0 and then administered therespective fusion protein on days 5, 7, and 9 (Table 8, experiment #1)or on days 5, 7, 9, and 11 (Table 8, experiment #2). These datareinforce the concept that IL2NAg fusion proteins are more tolerogenicthan IL4NAg fusion proteins and that IL2NAg fusion can suppress EAE whenadministered either before or after an encephalitogenic challenge.

M. Cytokine/NAg Fusion Proteins Target NAg to APC

As shown in FIG. 12, Panel A, purified cytokine/NAg fusion proteinsstimulated proliferation of an encephalitogenic CD4⁺ clone specific forthe 72-86 region of MBP in the presence of irradiated splenic APC. TheIL4.4 fusion protein was over 100-fold more potent than GPMBP based on acomparison of the antigen concentrations eliciting a half maximalresponse. The proliferative response to IL2.7 was bimodal.Concentrations of IL2.7 in the 10 pM to 1 nM range stimulatedapproximately 20,000 cpm of [³H]thymidine incorporation. IL2.7concentrations of 1 nM to 1 uM stimulated the second mode ofresponsiveness.

IL4.4 and IL2.7 stimulated T cell proliferation by a mechanismrestricted by MHCII I-A but not I-E (FIG. 12, Panel B). Responseselicited by IL4.4 and IL2.7 were completely abrogated by the OX6 mAb(anti-I-A MHCII) but were not affected by the OX17 (anti-1-E MHCII) mAb.Indeed, the antigenic reactivity of IL4.4 was inhibited by over 5 ordersof magnitude by OX6 whereas the response to GPMBP was inhibited by an˜100 fold margin. These findings were in accordance with the knownantigen restriction of the RsL.11 clone. Thus, when cultured with thesecytokine/NAg fusion proteins and irradiated splenic APC, RsL.11 T cellsexhibited antigen-stimulated proliferation rather than a mitogenicresponse to the cytokine domain. Other control cytokine/NAg fusionproteins (IL10.6, IL13.6, and NAgIL16-S) had antigenic reactivitysimilar to that of GPMBP (FIG. 12, Panel A). These responses were alsofully blocked by an anti-MHCII I-A mAb (OX6) and thereby representedantigenic responses to the encephalitogenic peptide (not shown). Thesedata indicated that the encephalitogenic GP73-87 sequence in each fusionprotein was processed and presented on MHCII glycoproteins.

Covalent linkage of the respective cytokine with the NAg was requiredfor enhanced potency of antigen recognition. IL4.4 was ˜100 fold morepotent than GPMBP even when GPMBP was added in culture with saturatingconcentrations of IL-4 as a separate molecule (FIG. 13, Panel A). IL-4activity of the IL4.4 protein and of the 1% and 0.1% IL4.4 baculovirussupernatants was confirmed in a mitogenesis assay of thymocytes (FIG.13, Panel C). Because thymocytes were costimulated with PMA andsaturating concentrations of IL-2 in all wells, the assay specificallydetected IL-4 activity but not IL-2 activity. The enhanced antigenicpotency of IL4.4 could not be explained by an independent action of IL-4on either APC or T cell responders. For example, the ability of IL-4 toinduce MHCII on B cells could not explain the enhanced potency of theIL4.4 antigen because IL4-induced MHCII induction would increaseantigenic potency without requirement for cytokine-antigen linkage. Theantigenic activity of IL4.4 was also substantially more active than theIL-4 activity of IL4.4 (compare FIG. 13, Panel A & FIG. 13, Panel C).

Covalent linkage of IL-2 and NAg was involved in the enhanced potency ofIL2.7 (FIG. 13, Panel B). That is, IL2.7 was ˜100 fold more potent thanGPMBP even when GPMBP was added to culture with saturatingconcentrations of IL-2 as a separate molecule. The IL-2 activity ofIL2.7 and the 1% and 0.1% IL2.7 baculovirus supernatants was confirmedin a mitogenesis assay of CTLL cells (FIG. 13, Panel D). IL-2 did notdirectly stimulate RsL.11 T cells because these T cells were rested andhad low concentrations of IL-2 receptors. Thus, the enhanced potency ofIL2.7 compared to GPMBP could not be explained by the mitogenic activityof IL-2. Rather, the covalent tethering of IL-2 and NAg allowedsynergistic antigenic activity that was not be duplicated by adding IL-2and NAg to culture as separate molecules.

The IL2.7 fusion protein was also substantially more potent than GPMBPin a T cell-mediated cytotoxic assay (FIG. 14). In these experiments,blastogenic MHCII⁺ T cells from the R1-trans T cell clone were APC.Previous studies showed that presentation of antigen by R1-trans T cellsto irradiated antigen-specific responders resulted in antigen-specifickilling of R1-trans T cells by a MHCII-restricted mechanism. In thisassay, R1-trans T cells grew rapidly in the presence of IL-2 unlessthese T-APC were killed by irradiated RsL.11 responders (14, 23). Theassays were devised such that IL2.7 fusion protein competed with IL-2for IL-2R on T-APC. Cultures were supplemented with rat IL-2 at theinitiation of culture (IL-2 at 0 hrs), 4 hrs after the initiation ofculture (IL-2 at 4 hrs), or 24 hrs after initiation of culture (IL-2 at24 hrs). R1-trans T-APC mediated rapid IL-2 dependent growth (y-axis)unless irradiated RsL.11 T cells killed R1-trans T cells uponrecognition of antigen on R1-trans T cells. Irradiation of RsL.11 Tcells prevented their antigen-specific proliferation but notcytotoxicity. The data indicated that when addition of IL-2 was delayeduntil 24 hours after the initiation of culture, IL2.7 was over 1000times more potent than GPMBP. However, when IL-2 was added at theinitiation of culture with IL2.7 or GPMBP, IL2.7 was only ˜32 fold morepotent than GPMBP. These data indicate that the IL2.7 fusion proteincompeted with IL-2 for cell surface IL-2 receptors and that thiscompetition determined the quantity of the IL2.7-associated NAg loadedinto the MHCII-antigen processing pathway. Overall, these data supportthe hypothesis that IL-2 receptors on T cells can be used to targetantigen to the MHCII-antigen processing pathway of activated T cells toenhance antigen recognition.

The potentiated responses of RsL.11 T cells to IL4.4 and IL2.7 that werestimulated in the presence of irradiated splenic APC were also inhibitedin part by IL-4 and IL-2, respectively. For example, IL-4 inhibited theresponse to IL4.4 by ˜10 fold but only slightly inhibited the responseto GPMBP (FIG. 15, Panel A) or IL2.7 (not shown). Likewise, IL-2inhibited the IL2.7 proliferative activity by ˜10 fold but slightlyenhanced proliferative responses to GPMBP (FIG. 15, Panel B) and IL4.4(not shown). These data provide additional evidence that the enhancedantigenic potency of IL4.4 and IL2.7 was due to targeting of fusionproteins to the respective cytokine receptors.

The availability of a monoclonal antibody (mAb) specific for IL-4 (OX81)provided another approach to assess the ability of the cytokine domainof the fusion protein to target the NAg to APC. This mAb allowedneutralization of the IL-4 domain in the IL4NAg fusion protein. Theantigenic activity of IL4.4 was inhibited by the OX81 mAb byapproximately 100-fold to the extent that IL4.4 was rendered equipotentto GPMBP (FIG. 16). The inhibitory action of OX81 was specific in thatOX81 did not affect antigenic responses stimulated by either GPMBP orIL2.7. These data indicate that in the presence of OX81, the NAg inIL4.4 had antigenic reactivity that was essentially equal to GPMBP andthat the encephalitogenic 73-89 sequences in IL4.4 and GPMBP wereprocessed and presented equally as measured by responses of clonedRsL.11 T cells.

N. Derivation and Expression of IL-16/NAg Fusion Proteins

Three different IL-16 expression systems were generated for this study:NAgIL16-S, IL16-S, and NAgIL16-L (Table 2). IL-16 is known to besynthesized as a large precursor protein that is cleaved by caspase-3into an N-terminal domain (˜1285 aa) that is translocated into thenucleus and a C-terminal domain (˜118 aa) that constitutes the activesecreted IL-16 (Zhang et al. (1998) J Biol Chem 273, 1144-1149; Zhang etal. (2001) J Biol Chem 276, 1299-1303). The NAgIL16-S, IL16-S, andNAgIL16-L fusion proteins were comprised of the 118 aa IL-16 C-terminaldomain.

Like the other cytokine/NAg fusion proteins described above (Tables 2and 9), the initial IL16NAg was expressed as a N-terminal IL-16 domainfused to a C-terminal EK-NAg-6his domain. However, this expressionsystem resulted in suboptimal antigenic activity. Hence, new IL16 werederived that positioned the NAg at the N-terminus and IL-16 at theC-terminus. Thus, the NAgIL16-S construction encoded the HBM signalsequence, a 7-histidine tag, the 69-87 sequence of GPMBP, and theC-terminal 118 aa fragment of IL-16. Primer design was based on themouse IL-16 sequence (Keane et al. (1998) J Immunol 160, 5945-5954)(accession AF006001). IL-16 cDNA was synthesized in the presence of 200nM of the 7H01 downstream primer, 1 μg total RNA, first strand buffer,dNTP, DTT, and SuperScript III Reverse Transcriptase (Invitrogen)according to manufacturer's instructions. To generate this fusionconstruct, an overlap extension reaction was used to generate theHBMss-7his-NAg DNA, and the IL-16 DNA was amplified in the same reactiontube. Primers were designed such that the HBMss-7his-NAg constructoverlapped the IL-16 DNA. Given the presence of excess upstream anddownstream primer, the full length HBMss-7his-NAg-IL16 constructresolved as a major product of this single PCR reaction. This reactionincluded the 3F09 upstream primer (200 nM) and the 3F12 primer (100 nM)to generate the N-terminal HBM-7his-GP69-87 construct together with the3F11 primer (100 nM) and the downstream 7H01 primer (200 nM) to amplifythe IL16 DNA. The reaction was catalyzed by Platinum High Fidelity TaqDNA Polymerase (Invitrogen). Cycling parameters included an initialdenaturation at 95° C. 5′ followed by 29 cycles of 94° C. 1′, 62° C. 2′,and 68° C. 2′, and then a final 68° C. 10′ extension step. The upstream3F09 and the downstream 7H01 primers included Spe I and Kpn Irestriction endonuclease sites that facilitated cloning of the constructinto the multiple cloning site of the pFastbac1 plasmid (Invitrogen).The recombinant NAgIL16-S pFastbac1 plasmid was then used for derivationof recombinant baculovirus as described in the BAC-TO-BAC BaculovirusExpression System (Invitrogen).

The IL16-S construct was generated by a similar overlap extension—PCRstrategy. The NAgIL16-S pFastbac1 plasmid was used as a template, andprimers (6H08 and 7H11) were designed to delete the sequence encodingthe GP69-87 NAg. The 3E06 upstream primer (200 nM) and the 6H08 primer(100 nM) were used to generate an N-terminal HBM-7his construct as anextension product, and in the same reaction tube, the 7H11 primer (100nM) and the 7H01 primer (200 nM) were used to amplify the IL-16 sequencefrom the plasmid. Due to overlap between these two constructs, the finalamplification product resolved as a contiguous HBMss-7his-IL16 constructflanked by Spe I and Kpn I restriction endonuclease sites. Thisconstruct was inserted into the pFastbac1 vector, which was used toderive the IL16-S recombinant baculovirus.

Publication of the predicted rat IL-16 sequence (XP_(—)218851) revealedthat the C-terminal amino acid accounted for the only difference betweenthe predicted rat sequence (C-terminal leucine) and either NAgIL16-S orIL16-S(C-terminal serine). To express authentic rat IL-16 (C-terminalleucine), site-directed mutagenesis was used to alter this codon. Togenerate the NAgIL16-L construct, the NAgIL16-S pFastbac1 plasmid wasused as template for a whole plasmid PCR in the presence of the 7A01 and1F07 primers and Platinum Pfx DNA polymerase. 7A01 was the mutagenicprimer containing a 2-nucleotide substitution (TTG substituted in placeof for TCA) in the terminal 5′ codon. This primer and the 1F07 primerwere overlapping primers that generated linearized copies of theplasmid. The reaction mix was melted at 95° C. for 5′ followed by 29cycles at 94° C. for 30″, 60° C. for 1′, 68° C. for 3′ followed by a 68°C. 10′ extension step. The products were washed and concentrated by useof Microcon YM100 Centrifugal Filter Devices, were treated with Dpn Irestriction endonuclease to deplete residual parental plasmid, and wereused to transform Top10 E coli by electroporation.

Upstream primers contained a Spe I site (ACTAGT) and downstream primerscontained a Kpn I site (GGTACC) to facilitate cloning of fusion insertsinto the multiple cloning site of the pFastBac1 vector (Table 3). TheDNA constructs were cut with the combination of KpnI and SpeIrestriction endonucleases and were ligated into (Kpn I×Spe I)-cutpFastbac1 vector (Invitrogen). Recombinant plasmids were transformedinto Top10 E. coli. Plasmid DNA was purified and sequenced to confirmthe predicted sequence. Recombinant plasmids were then transformed intoDH10Bac E. coli to achieve transposition of the fusion construct intothe baculovirus genome. Baculovirus DNA was purified and transfectedinto Sf9 insect cells to generate supernatants containing recombinantbaculovirus. Supernatants containing the recombinant fusion proteinswere confirmed by PCR and by secretion of the predicted protein intosupernatants of infected Sf9 cells.

O. Purification of Cytokine/NAg Fusion Proteins

Purification of proteins bearing a C-terminal 6-histidine tag includingIL1RA/NAg4, IL2.7, IL2Ekdel, IL2, IL10.6, and IL13.6 were describedpreviously. Proteins bearing an N-terminal histidine tag (NAgIL16-S,IL16-S, and NAgIL16-L) were purified by immobilized metal ion affinitychromatography (IMAC) on Ni resin (Qiagen). Fusion proteins weresecreted into the supernatant by Sf9 insect cells infected with therespective baculovirus and were concentrated on YM10 or YM5ultrafiltration membranes. Concentrated supernatants were loaded onto 1ml beds of Ni resin, were extensively washed (50 mM NaH₂PO₄, 500 mMNaCl, 10 mM imidazole, pH 8.0) and then eluted with 250 mM imidazole (pH8.0). Proteins were then concentrated and reapplied to a second step ofIMAC. Protein quantity was assessed by the BCA protein assay (Pierce),and purity was assessed by SDS-PAGE.

P. Tolerance Induction

To determine whether cytokine/NAg proteins prevent active induction ofEAE in Lewis rats, rats were given 3 subcutaneous injections of a givencytokine/NAg protein (1 nmole dose) in saline at 1-2 week intervals asdesignated. At least 7 days after the last injection of the respectivefusion protein, rats were challenged with NAg in CFA (day 0) to induceEAE. Alternatively, cytokine/NAg fusion proteins were tested todetermine whether these proteins could ameliorate EAE when administeredafter challenge.

Q. Induction and Clinical Assessment of EAE

EAE was induced in Lewis rats by injection of an emulsion containingGPMBP in CFA (200 μg Mycobacterium tuberculosis). In designatedexperiments, rats were challenged with the DHFR-NAg fusion protein. Thisprotein was comprised of the mouse dihydrofolate reductase as theN-terminal domain and the encephalitogenic GP69-87 peptide of GPMBP asthe C-terminal domain (manuscript in preparation). The following scalewas used to assign intensity of EAE; paralysis in the distal tail, 0.25;limp tail, 0.5; ataxia, 1.0; hind leg paresis, 2.0; full hind legparalysis, 3.0. The cumulative score for each rat consisted of the sumof daily scores for each animal. The mean cumulative score for a groupwas calculated by averaging the cumulative scores for all rats within agroup. The mean maximal intensity scores were assigned to each groupbased on the average maximum score among afflicted rats within a group.The mean severity of EAE (y-axis of graphs) was the average score of allrats in a group on a given day.

R. Cytokine/NAg Fusion Proteins

The baculovirus expression systems for cytokine/NAg fusion proteinsIL1RANAg, IL2NAg, IL4NAg, IL10NAg, IL13NAg were described previously.These fusion proteins had predicted cytokine activities as assessed bybioassay. The IL1RA/NAg4 protein blocked the ability of IL-1 to enhanceproliferation of Con A-stimulated thymocytes without inhibiting ConA-stimulated thymocyte proliferation in the absence of IL-1. IL2NAgstimulated proliferation of rat splenic T cells, thymic T cells, andestablished T cell lines. IL4NAg stimulated proliferation ofPMA-costimulated thymocytes but inhibited the IL-2 dependent growth ofMBP-specific and conalbumin-specific T cell clones. Both IL4NAg andIL13NAg blocked γIFN-induced nitric oxide production by macrophages.IL10NAg enhanced the IL-2 dependent growth of established T cell clones.

S. Comparative Tolerogenic Activity of Cytokine/NAg Fusion Proteins

NAgIL16 and NAgIL2 fusion proteins were injected subcutaneously insaline at a dose of 1 nmole every 1-2 weeks for a total of threeinjections. Two different forms of NAgIL16 (NAgIL16-S and NAgIL16-L) andtwo different forms of IL2NAg (IL2.7 and IL2Ekdel) were tested fortolerance induction. Seven days after the last injection ofcytokine/NAg, rats were actively challenged with GPMBP or with DHFR-NAg.As shown in Table 9, both NAgIL16 and IL2NAg had tolerogenic activity.Both versions of NAgIL16 were highly effective tolerogens andsignificantly reduced the mean cumulative score of EAE compared to ratspretreated with equal doses of NAg (GPMBP or GP69-88 peptide). NAgIL16also significantly decreased the mean maximal intensity and delayed theonset of EAE. Although IL2.7 and IL2Ekdel were not as efficient asNAgIL16, both IL2NAg proteins were effective tolerogens compared toGPMBP or the synthetic peptide GP69-88. IL2NAg significantly reduced themean cumulative score and mean maximal intensity of EAE andsignificantly delayed onset of disease. Thus, two independently derivedversions of NAgIL16 and two independently derived versions of IL2NAg hadtolerogenic activity.

Other anti-inflammatory or tolerogenic fusion proteins were tested toaddress the question of whether NAgIL16 and IL2NAg fusion proteinsexhibited unique tolerogenic activity (Table 10). Again, the fusionproteins were administered in three injections (days −21, −14, & −7, 1nmole/injection), and 7 days were allowed to elapse between the lastpretreatment and the encephalitogenic challenge (50 μg GPMBP in CFA).The IL10.6 did not exhibit tolerogenic activity whereas the IL13.6 andIL1RA/NAg4 fusions proteins decreased the mean cumulative score and meanmaximal activity and delayed disease onset compared to rats pretreatedwith saline. However, these measures were not significant compared tothose of rats pretreated with GPMBP. Overall, the rank order oftolerogenic activity for the six fusion proteins in the Lewis rat modelof EAE was: NAgIL16>IL2NAg>>IL1RANAg, IL13NAg≧IL10NAg, GP69-88,GPMBP≧saline (Tables 9 and 10).

T. Linkage of IL-16 and NAg was Indicated in Tolerance

The tolerogenic activity of the NAgIL16 fusion proteins was contingentupon the covalent linkage of IL-16 and NAg (Table 11). Rats pretreatedwith NAgIL16 were resistant to the subsequent active induction of EAE.These rats showed a significant reduction in the mean cumulative scoreand mean maximal intensity together with a delayed onset of disease.Furthermore, rats pretreated with NAgIL16 did not exhibit a spontaneousrelapse of EAE. In contrast, rats pretreated with a combination of IL16and GP69-88 as separate molecules or with either agent alone were fullysusceptible to EAE, and many of these rats showed a spontaneous relapseof disease. These data indicated that tolerance induction reacted to thelinked activity of IL-16 and NAg. Thus, the mechanism of toleranceinduction was antigen-specific rather than an antigen-nonspecificcytokine-mediated effect. Furthermore, the tolerogenic activity elicitedby NAgIL16 endured long after the last administration of the fusionprotein.

Example 3 Tolerogenic Vaccines

Because immunological tolerance is antigen-dependent, a pretreatmentregimen was best suited for finding an antigen-specific tolerogenicvaccine. Conversely, treatment regimens whereby fusion proteins wereadministered during or after encephalitogenic sensitization may revealthe immunosuppressive activities of the anti-inflammatory cytokinedomain and may not involve an antigen-dependent tolerogenic response.Experiments were performed to determine whether these fusion proteinswould inhibit EAE when administered during the sensitization or effectorphase of EAE or whether these fusion proteins may augment EAE or haveunanticipated allergic or anaphylactic activities.

The NAgIL16 fusion protein was highly effective when deliveredsystemically at the onset of the effector phase (Table 12 and FIG. 17).Rats challenged on day 0 with DHFR-NAg in CFA were treated with 5 nmolesNAgIL16-L intravenously on day 8 and then were given an equivalentintraperitoneal injection on day 12. These injections completely blockedprogression to severe EAE. Compared to control rats that receivedequivalent dosages of GP69-88, rats receiving NAgIL16-L exhibited asignificant reduction in the mean cumulative score and the mean maximalintensity and showed a delayed onset of disease. These data indicatethat NAgIL16 regulates events that are involved in the effector phase ofEAE.

Systemic administration may provide sufficient bioavailability, suchthat the fusion protein may interact with effector cells that havedisseminated from lymph nodes to the spleen and CNS.

Subcutaneous administration of NAgIL16 during the effector phase alsoreduced the severity of EAE (Table 13). Pre-challenge treatment regimens(Tables 5-11) showed that NAgIL16 and IL2NAg were the most effectivetolerogens compared to the other cytokine/NAg fusion proteins or to NAgalone. Likewise, this post-challenge treatment regimen (administrationof cytokine/NAg on days 5, 7, and 9) revealed the same finding—thatNAgIL16 and IL2NAg were the most effective for inhibition of EAE.Treatment with IL2NAg or NAgIL16 significantly reduced the meancumulative score and mean maximal intensity of EAE (Table 13). IL4Ekdel,IL10.6, IL13.6, and IL1RA/NAg4 were administered to parallel groups andwere less effective inhibitors. Subcutaneous injections wereadministered at sites adjacent to the encephalitogenic challenge so thatthe fusion protein would enter the same lymphatic drainage. These dataindicate that the two fusion proteins exhibiting the greatesttolerogenic potency in pretreatment regimens were also the two mosteffective proteins for attenuating the effector phase of EAE. NAgIL16and IL1RA/NAg4 were also tested in a regimen to deliver the fusionprotein subcutaneously throughout the sensitization phase (days −1, 3,and 7) of EAE (Table 14). Again, NAgIL16 reduced the severity of EAEwhereas IL1RA/NAg4 was less effective. The ability of NAgIL16 to inhibitthe effector phase of EAE was antigen-dependent (Table 15). The fusionprotein NAgIL16 reduced the mean cumulative score, the mean maximalintensity, and decreased the incidence of severe EAE whereasadministration of IL-16 and NAg as separate molecules was ineffectiveand did not alter the course of disease.

To test the effect of dosage for NAgIL16-mediated tolerance, rats werepretreated with NAgIL16 at four different doses (FIG. 18). The data showthat the ability of NAgIL16 to induce tolerance was dose dependent. Thehighest two doses elicited the most profound tolerogenic response.

The NAgIL16 fusion protein was the most effective tolerogen. This fusionprotein decreased severity of EAE and decreased the incidence of severeEAE. Both pre-challenge and post-challenge delivery regimens wereeffective. When a pre-challenge delivery regimen was combined with apost-challenge delivery regimen (Table 16), the NAgIL16 alsosignificantly suppressed disease incidence to 22% (compared to 100% inthe two respective control groups). These data indicate that morechronic treatment regimens have the potential to completely inhibit aparalytic demyelinating autoimmune disease in rodents.

Example 4 Fusion Proteins Target the MHCII Antigen Processing Pathway

A. IL2NAg

IL2NAg may target the covalently-tethered NAg into the majorhistocompatibility complex class II (MHCII) antigen processing pathwayof activated T cells.

First, purified cytokine/NAg fusion proteins stimulated antigenicproliferation of an encephalitogenic CD4⁺ clone specific for the 72-86region of MBP in the presence of irradiated splenic APC. Theproliferative response to IL2NAg was bimodal (data not shown).Concentrations of IL2NAg in the 10 pM to 1 nM range stimulatedapproximately 20,000 cpm of [³H]thymidine incorporation. At thisconcentration range, IL2.7 was substantially more active than GPMBP. Inthis assay, IL4NAg also exhibited enhanced antigenic potency. IL4NAg was˜100 fold more active than GPMBP. Other control cytokine/NAg fusionproteins (IL10NAg, IL13NAg, and NAgIL16) had antigenic reactivitysimilar to that of GPMBP. These fusion proteins stimulated T cellproliferation by a mechanism restricted by MHCII I-A but not 1-E. Thesedata indicated that the encephalitogenic GP73-87 sequence IL2NAg wasprocessed and presented on MHCII glycoproteins.

Second, covalent linkage of IL-2 with the NAg was required for enhancedpotency of antigen recognition of IL2NAg. IL2.7 was ˜100 fold morepotent than GPMBP even when GPMBP was added to culture with saturatingconcentrations of IL-2 as a separate molecule. The IL-2 activity ofIL2.7 and the 1% and 0.1% IL2 baculovirus supernatants was confirmed ina mitogenesis assay of CTLL cells. IL-2 did not directly stimulateRsL.11 T cells because these T cells were rested and had lowconcentrations of IL-2 receptors. Thus, the enhanced potency of IL2.7compared to GPMBP could not be explained by the mitogenic activity ofIL-2. Rather, the covalent tethering of IL-2 and NAg enabled synergisticantigenic activity that could not be duplicated by adding IL-2 and NAgto culture as separate molecules. The most consistent interpretation isthat the cytokine domain interacted with the respective cytokinereceptor on APC to target the covalently linked NAg into the MHCIIantigen processing pathway.

Third, IL4NAg was ˜100 fold more potent than GPMBP even when GPMBP wasadded in culture with saturating concentrations of IL-4 as a separatemolecule. IL-4 activity of the IL4.4 protein and of the 1% and 0.1% IL4baculovirus supernatants was confirmed in a mitogenesis assay ofthymocytes. Because thymocytes were costimulated with PMA and saturatingconcentrations of IL-2 in all wells, the assay specifically detectedIL-4 activity but not IL-2 activity. The enhanced antigenic potency ofIL4.4 could not be explained by an independent action of IL-4 on eitherAPC or T cell responders. For example, the ability of IL-4 to induceMHCII on B cells could not explain the enhanced potency of the IL4.4antigen because IL4-induced MHCII induction would increase antigenicpotency without requirement for cytokine-antigen linkage.

Fourth, the IL2NAg fusion protein was also more potent than GPMBP in a Tcell-mediated cytotoxic assay. The enhanced potency of IL2NAg wasreversed by the addition of IL-2 at the initiation of culture. When IL-2was added at the initiation of culture, IL2NAg was only ˜32 fold morepotent than GPMBP. When the addition of IL-2 was delayed until 24 hoursafter the initiation of culture, IL2NAg was over 1000 times more potentthan GPMBP. Thus, IL2NAg competed with IL-2 for cell surface IL-2receptors, and this competition determined the amount ofIL2NAg-associated NAg loaded into the MHCII-antigen processing pathwayof these activated T cells. Overall, these data support the hypothesisthat IL-2 receptors on activated T cells can be used to target antigento the MHCII-antigen processing pathway of activated T cells to enhanceantigen recognition

Fifth, the potentiated responses of RsL.11 T cells to IL4NAg and IL2NAgthat were stimulated in the presence of irradiated splenic APC were alsoinhibited in part by IL-4 and IL-2, respectively. For example, IL-4inhibited the response to IL4NAg by ˜10 fold but only slightly inhibitedthe response to GPMBP. Likewise, IL-2 inhibited the IL2NAg stimulatedproliferative activity by ˜10 fold but slightly enhanced proliferativeresponses to GPMBP. Lastly, the potentiated responses of RsL.11 T cellsto IL4NAg were specifically blocked in the presence of the OX81 anti-IL4mAb. These data provide additional evidence that the enhanced antigenicpotency of IL4NAg and IL2NAg was due to targeting of fusion proteins tothe respective cytokine receptors.

B. NAgIL16

NAgIL16 may target the covalently-tethered NAg into the MHCII antigenprocessing pathway of activated T cells. NAgIL16 was targeted forpresentation by nonadherent Con A-activated splenic T cells (FIG. 19).Lewis rat splenocytes were cultured for 2 days in complete RPMI medium.Nonadherent splenocytes were then cultured with 100 nM, 10 nM, 1 nM, or100 pM of designated fusion proteins in the presence of 2.5 μg/ml Con A.After 3 days of culture, splenic lymphoblasts were washed, irradiated,and used at designated cell densities as APC (x-axis) to stimulate theGPMBP-specific RsL.11 T cell line. Thus, antigen was incorporated intoMHC class II glycoproteins during a 3-day culture in the presence of ConA, and then antigen presentation was measured in a subsequent 3-dayculture in the absence of soluble antigen. The results showed that aconcentration of 1 nM NAgIL16-L elicited stronger antigen presentationthan 10 nM GPMBP or 100 nM DHFR-NAg. These data indicated that the IL16moiety potentiated antigen presentation by at least 10-fold compared toNAg alone.

C. IFNβNAg

The cytokine domain of IFNβNAg has potent cytolytic activity for IL-2activated T cells. The IFNβ domain endows APC with cytotoxic activityand simultaneously loads the covalently tethered NAg into the MHCIIantigen processing compartment. The relevant APC in this model may ormay not be T cells. This mechanism would deplete NAg-specific T cellresponders and render rats resistant to EAE.

Example 5 In Vitro Activity of Purified IFNβNAg and IL2NAg FusionProteins

The IFNβNAg & IL2NAg fusion proteins exhibited potent biologicalactivity (FIG. 20). The IL2NAg fusion proteins were active in the lowpicomolar range (half maximal responses in the 3-10 pM in standard CTLLassays). The IFNβNAg also exhibited a potency in the 1-10 pM range in aT cell cytotoxicity assay. In this assay, IFNβNAg directly killed IL-2stimulated T cells (either the BN-GP T cell clone or the RsL.11 T cellclone) during a 3 day culture. These potencies compare very favorably tocommercial IL-2 and IFNβ preparations.

Example 6 Cytokine/NAg Effects on Ongoing Autoimmune Encephalomyelitis

The results from an experiment in which in vivo tolerogenic activitiesof the NAgIL16 fusion protein in the Lewis rat model of Autoimmuneencephalomyelitis (EAE) EAE are summarized in Tables 12 and 15. Thesedata indicate that NAgIL16 can also reverse an ongoing encephalitogenicimmune response and that this inhibitory activity is specific for NAg.

Example 7 NAgIL16 Treatment Inhibits Progression of EAE after DiseaseOnset

Administration of NAgIL16 during clinical disease halted EAE progressionand prevented subsequent relapse as shown in FIG. 21. Data presented intabular form (Table 19, Experiment 2) are shown as a time course of EAE.On day 11, rats were matched for clinical signs of EAE and were randomlyassigned to one of two groups that were injected with either NAg(GP69-88) or NAgIL16L. (A) Treatments were on day 11 (5 nmoles in salinei.v.), day 12 (5 nmoles in saline i.p.), and day 14 (2 nmoles in salinei.v.) (see arrows). The cumulative EAE severity per day forNAgIL16-treated rats was significantly less than that for NAg-treatedrats on days 13, 13.5, and 14 (p=0.013, 0.005, and 0.009, respectively).The mean cumulative scores (tallied after the first treatment on day 11)also were significantly different (p<0.001) (Mann-Whitney Test). (B) Thefrequency of relapses (onset after day 21) for NAg-treated rats (6 of 7,85.7%) was significantly higher than for NAgIL16-treated rats (0 of 7)(p=0.0047, Fisher's Exact Test).

Thus, NAgIL16 had an acute inhibitory effect that suppressed an ongoingencephalitogenic attack and facilitated recovery. NagIL16 also exhibitedlonger term tolerogenic activity that prevented a subsequent relapse.

Example 8 Covalent Tethering of IL-2 and NAg in IL2NAg-Induced EAEInhibition During the Onset of EAE

The covalent linkage of IL2 and NAg played a role in theimmunosuppressive activity of the post-challenge treatment regimen(Table 20). IL2NAg substantially reduced the median cumulative score,the median maximal intensity, the incidence of severe EAE, and the meannumber of days that rats were afflicted with severe EAE whereas deliveryof IL-2 and NAg simultaneously as separate molecules had no tolerogenicactivity. These data indicate that IL2NAg is a highly effectiveinhibitor when delivered after encephalitogenic challenge and thatIL2NAg can attenuate an ongoing encephalitogenic immune response.Comparison of the post-challenge treatment regimens (days 5, 7, & 9;days 5, 7, 9, & 11; days 5, 7, 9, 11, & 13) for data shown in Table 20see above showed progressively more effective inhibition of EAE astreatments were extended into the phase of disease onset and maximalparalysis. These data provide suggestive evidence that IL2NAg directlyimpairs the effector phase of an ongoing encephalitogenic immuneresponse.

Example 9 Dosing Regimens

Optimal doses that inhibit the subsequent induction of EAE areestablished. A dose of 1 nmole per injection (divided as 0.5 nmole oneach side of the base of the tail) with a total of three injections isgiven at 1-2 week intervals. Dosing of fusion protein and induction oftolerance is shown by subcutaneous injection of NAgIL16-L, IFNβ.4, orIL2.7 by a regimen (total of 3 injections, one injection each on day−21, −14, and −7 followed by an encephalitogenic challenge with 50 μgDHFR-NAg on day 0). Groups of rats (n=10) receive 100 pmoles, 320pmoles, 1 nmole, or 3.2 nmoles of each cytokine/NAg. One additionalgroup of rats (n=4) receives 10 nmoles cytokine/NAg protein.

Higher doses of cytokine/NAg will either induce more profound tolerance,or induction is dependent upon low dose administration of thecytokine/NAg and that a high dose may be neutral or may promotesensitization as indicated by acceleration or more intense disease afterchallenge. High dose administration provides information regardingsafety of each cytokine/NAg. At 1 nanomole doses, these fusion proteinsdid not result in a local reaction or any indication of EAE induction.Establishment of dose-response curves reveals an optimal dose as abalanced consideration of efficacy versus practicality of generating thegiven dosage. If high dose administration promotes any aspect of EAEinduction, then using low doses exclusively or an escalating dosestrategy similar to those currently used in the clinic for allergicdesensitization are considered.

Dose-Dependence of NAgIL16-Mediated Inhibition of EAE

Cytokine/antigen fusion proteins may elicit a balance of effector andregulatory cells, and this balance may vary with dosage. Tolerogenicfusion proteins would predictably cause tolerance by dose—dependentmechanisms. Higher doses should induce more profound tolerance. However,paradoxical dosage effects are possible, and higher doses may moreefficiently prime effector cells and promote immunity. To test theeffect of dosage for NAgIL16-mediated tolerance, rats were pretreatedwith NAgIL16 at four different doses (FIG. 18). Rats were injected withsaline or with 0.5, 1.0, 2.5, or 5.0 nmoles NAgIL16 on days −21, −14,and −7 and then were challenged with 50 μg DHFR-NAg in CFA on day 0. Allfour dosages of NAgIL16 resulted in significant tolerance, and the twohighest two doses elicited the most profound tolerogenic response. Thesedata indicate that the tolerogenic activity of NAgIL16 was dosedependent and that optimal tolerance may be induced with doses of2.5-5.0 nmoles.

Example 10 Combination Treatments

NAgIL16 and IL2NAg fusion proteins can act synergistically. IFNβ mayfacilitate expansion of regulatory T cell subsets. Hence, thecombination of IFNβNAg and either NAgIL16 or IL2NAg can load NAg intothe MHCII antigen processing pathway of regulatory T cells.

Rats were immunized with 50 μg of DHFR-NAg in CFA on day 0. Rats(cytokine/NAg group) were injected subcutaneously with 1 nmole IL2/NAg(IL2Ekdel) in saline on days 5, 7, 10, and 12 and were also injectedintravenously with 5 nmoles NAgIL16 in saline on days 8 and 11.

The experiment shown in Table 21 verified the efficacy of a combinedtreatment protocol including the use of IL2NAg and NAg IL6 fusionproteins injected on alternative days into the same group of rats. Thiscombined treatment protocol eliminated severe paralytic disease andminimized disease to the distal tip of the tail. In contrast, controlrats exhibited a full course of paralytic EAE. These data indicate thattwo fusion proteins (IL2NAg and NAgIL16) can be used in a combinedtreatment protocol to control an encephalitogenic attack.

Example 11 NAgIL16-Induced Tolerance

These experiments provide a balanced experimental design that assess theantigen specificity of the tolerogenic pathway, as opposed to adisease-specific or organ-specific mechanism or an antigen nonspecificcytokine-dependent mechanism of tolerance.

The mechanism by which the NAgIL16 fusion protein induced tolerance wasaddressed by adoptive transfer experiments discussed in Table 20. Theprediction was that a mechanism of active dominant tolerance would betransferred from tolerant donors to naïve recipients by the transfer oflymphoid cells. In contrast, a passive mechanism of tolerance could notbe transferred from tolerant donors to naïve recipients.

Rats were pretreated with 4 nmoles of GP69-88 (NAg) or 4 nmoles NAgIL16in saline on days −28, −21, and −14. On day −7, draining lymph nodecells and splenocytes were pooled from rats of each group and wereinjected (i.p.) into recipient rats (1:1 donor to recipient ratio).Seven days after adoptive transfer, recipient rats were challenged onday 0 with 50 μg of DHFR-NAg in CFA.

Table 22 provides evidence that sensitization of rats with NAgIL16 insaline promotes a mechanism of active tolerance as assessed by adoptivetransfer. Donor rats were pretreated with 4 nmoles of NAgIL16 in salineor with 4 nmoles of the encephalitogenic peptide GP69-88 (NAg) in salineon days −28, −21, and −14. Sensitized splenocytes and draining lymphnode cells from donor rats were harvested on day −7 and were transferredto recipients without culture. Seven days later, on day 0, recipientrats were challenged with DHFR-NAg in CFA to induce EAE. The resultssuggest that the NAgIL16 fusion protein elicited a dominant mechanism ofactive tolerance.

TABLES

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCT Cysteine Cys CTGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu E GAA GAGPhenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGG GGT Histidine HisH CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys K AAA AAG Leucine LeuL TTA TTG CTA CTC CTG CTT Methionine Met M ATG Asparagine Asn N AAC AATProline Pro P CCA CCC CCG CCT Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGT Serine Ser S AGC ACT TCA TCC TCG TCT ThreonineThr T ACA ACC ACG ACT Valine Val V GTA GTC GTG GTT Tryptophan Trp W TGGTyrosine Tyr Y TAC TAT

TABLE 2 Design of cytokine/NAg fusion proteins. Descriptor^(a) Name^(b)N to C terminal order of domains^(c) IL1RANAg IL1RA/NAg4 nativess-IL1RA-EK-(GP73-87)-6his IL1RAwoNAg IL1RA native ss-IL1RA--6his IL2NAgIL2.7 native ss-IL2-EK-(GP73-87)-6his IL2NAg IL2Ekdel nativess-IL2-(GP73-87)-6his IL2woNAg IL2-D native ss-IL2-6his IL2woNAg IL2native ss-IL2 IL4NAg IL4.4 native ss-IL4-EK-(GP73-87)-6his IL4NAgIL4Ekdel native ss-IL4-(GP73-87)-6his IL4woNAg IL4-A native ss-IL4-6hisIL4woNAg IL4 native ss-IL4 IL10NAg IL10.6 nativess-IL10-EK-(GP73-87)-6his IL10woNAg IL10 native ss-IL10-EK-6his IL13NAgIL13.6 native ss-IL13-EK-(GP73-87)-6his IL13woNAg IL13-A nativess-IL13-6his IL13woNAg IL13-B native ss-IL13-6his IFN□ IFN□.4 nativess-IFN□-EK-(GP73-87)-6his IFN□woNAg IFN□ native ss-IFN□-6his NAgIL16^(d)NAgIL16-S HBM ss-7his-NAg-C-terminal IL16 IL16 alone^(d) IL16-S HBMss-7his-C-terminal IL16 NAgIL16^(d) NAgIL16-L HBM ss-7his-NAg-C-terminalIL16 ^(a)The descriptor defines the presence and the relativeorientation of the cytokine and NAg domains in the fusion protein.^(b)The name refers to the specific names given to specific proteins.^(c)N to C terminal order of domains provides for a more detaileddescription of the relative order of domains for each recombinantprotein. For example, in IL1RA/NAg4, the protein consisted of the nativeIL1RA signal sequence (ss), the mature IL-1RA cytokine, a enterokinase(EK) domain (G-D-D-D-D-K-G, SEQ ID NO:1), the major encephalitogenicpeptide of GPMBP (P-Q-K-S-Q-R-S-Q-D-E-N-P-V-V-H, SEQ ID NO:2), and a6-his C-terminal tag. The IL2.7, IL4.4, IL10.6, IL13.6, and IFNβ.4fusion proteins had a parallel structural design comprised of anN-terminal cytokine domain and a C-terminal EK-NAg-6his domain. Theselatter fusions also had the native ss of the respective cytokine. In theIL1RAwoNAg, IL2woNAg, IL4woNAg, IL10woNAg, IL13woNAg, and IFNβwoNAgproteins, the cytokine was expressed without the Ek-NAg domain. RatIL2Ekdel and IL4Ekdel had a deletion of the EK domain but were otherwiseidentical to IL2.7 and IL4.4 respectively. Rat IL2 and IL4 lacked theEK-NAg domain and were the only proteins lacking a C-terminal 6his tag.^(d)NAgIL16-S and IL16-S were cloned before the rat IL-16 sequence wasavailable, and the cloning was accomplished by use of primers based onthe mouse IL-16 sequence. These proteins have a C-terminal serine of themouse sequence whereas the remainder of the protein encompasses thenative rat sequence. The native C-terminal amino acid in rat IL-16 is aleucine. NAgIL16-S protein consisted of the honey bee mellitin (HBM)signal sequence (M-K-F-L-V-N-V-A-L-V-F-M-V-V-Y-I-S-Y-I-Y-A, SEQ IDNO:34), a 7-histidine tag, the encephalitogenic 69-87 peptide of GPMBP(Y-G-S-L-P-Q-K-S-Q-R-S-Q-D-E-N-P-V-V-H, SEQ ID NO:35), and theC-terminal 118 aa sequence of IL-16 with a C-terminal serine. NAgIL16-Lwas identical to NAgIL16-S except for the C-terminal substitution ofleucine. IL16-S had a modified HBM ss(M-A-F-L-V-N-V-A-L-V-F-M-V-V-Y-I-S-Y-I-Y-A, SEQ ID NO:36), a 7-histidinetag, and the C-terminal 118 aa sequence of IL-16 with a C-terminalserine.

TABLE 3 Primers used to construct genes encoding cytokine/NAg fusionproteins. Primer name (code) Sequence of primer* 5′ IL-1RA (3B05)ATAACTAGTATGGAAATCTGCAGGGGACCTTACAGTCAC (SEQ ID NO:5) 5′ IL-2 (3B12)ATAACTAGTATGGCCTACAGCATGCAGCTC (SEQ ID NO:6) 5′ IL-4 (2D11)ATAACTAGTATGGGTCTCAGCCCCCACCTTGCTG (SEQ ID NO:7) 5′ IL-10 (2D12)ATAACTAGTATGGCACTTGGCTCAGCACTGCTATGTT (SEQ ID NO:8) 5′ IL-13 (2E01)ATAACTAGTATGGCACTCTGGGTGACTGCAG (SEQ ID NO:9) 3′ IL-1RA (5A02)CCTTTGTCATCGTCATCACCTTGGTCTTCCTGGAAGTAGAA (SEQ ID NO:10) 3′ IL-2 (5A03)CCTTTGTCATCGTCATCACCCTGAGTCATTGTTGAGATGAT (SEQ ID NO:11) 3′ IL-4 (5A04)CCTTTGTCATCGTCATCACCGGACATGGAAGTGCAGGACTG (SEQ ID NO:12) 3′ IL-10 (5A05)CCTTTGTCATCGTCATCACCATTTTTCATTTTGAGTGTCAC (SEQ ID NO:13) 3′ IL-13 (5A06)CCTTTGTCATCGTCATCACCGTGGCCATAGCGGAAAAGTTG (SEQ ID NO:14) 5′ EK-NAg(5A01) GATGACGATGACAAAGGACCCCAGAAGTCGCAGCGGTCCCAAG (SEQ ID NO:15) 3′ NAg(2F07)TATGGTACCTTAGTGATGGTGATGGTGATGGACTACAGGGTTTTCATCTTGGGACCGCTGCGACT (SEQ ID NO:16)IL2EKdel (5H04)ATCATCTCAACAATGACTCAG\CCCCAGAAGTCGCAGCGGTCCCAA (SEQ ID NO:17) IL2EKdel(6E12) CTGAGTCATTGTTGAGATGATGCTTTGACAGAT (SEQ ID NO:18) IL4EKdel (5H05)CAGTCCTGCACTTCCATGTCC\CCCCAGAAGTCGCAGCGGTCCCAA (SEQ ID NO:19) IL4EKdel(6F01) GGACATGGAAGTGCAGGACTGCAAGTATTTCCCTCGT (SEQ ID NO:20) 3′ IL2-D(6H03)TATGGTACCTTAGTGATGGTGATGGTGATG\CTGAGTCATTGTTGAGATGATGCT (SEQ ID NO:21)3′ IL4-A (6H04)TATGGTACCTTAGTGATGGTGATGGTGATG\GGACATGGAAGTGCAGGACTGCA (SEQ ID NO:22) 3′IL13-A (6H06)TATGGTACCTTAGTGATGGTGATGGTGATG\GTGGCCATAGCGGAAAAGTTGCTT (SEQ ID NO:23)5′ HBM7hNAg (3F12)GACCGCTGCGACTTCTGGGGCAGGGAGCCATAATGGTGATGGTGATGGTGATGGGCATAGATGTAAGAAATGTA(SEQ ID NO:24) 3′ HBM7hNAg (3F09)ATAACTAGTATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCC (SEQ ID NO:25)5′ NAgIL16S (3F11)TCCCTGCCCCAGAAGTCGCAGCGGTCCCAAGATGAAAACCCTGTAGTCCATTCTGCTGCATCAGCTTCAGTA (SEQ ID NO:26)3′ NAgIL16S (7H01) TATGGTACCTTATGAGTCTGCAGAAGCTGTTGTCTG (SEQ ID NO:27)5′ HBM7h (3E06)ATAACTAGTATGGCATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACAT (SEQ ID NO:28)3′ HBM7h (6H08)GCAGAATGGTGATGGTGATGGTGATGGGCATAGATGTAAGAAATGTATACGACCATAAAAAC (SEQ ID NO:29)5′ IL16S (7H11)CATCACCATCACCATCACCATTCTGCTGCATCAGCTTCAGTAGC (SEQ ID NO:30) NAgIL16Lmut(7A01) GACAACAGCTTCTGCAGACTTGTAAGGTACCAAGCTTGTC (SEQ ID NO:31) NAgIL16L(1F07) AGTCTGCAGAAGCTGTTGTCTGCTTGCCCT (SEQ ID NO:32) *Upstream primerscontained a Spe I site (ACTAGT) and downstream primers contained a Kpn Isite (GGTACC) to facilitate cloning of fusion inserts into the multiplecloning site of the pFastBac1 vector. These restriction sites areunderlined. The \ symbol shown for the 5H04 and 5H05 primers representsthe juncture for deletion of the EK encoding region. The \ symbol shownfor the 6H03, 6H04, and 6H06 primers represents the juncture fordeletion of the EK-NAg encoding region. Overlap extension PCR was usedto generate many of these constructs. For example, the NAgIL16-Sconstruction encoded the HBM signal sequence, a 7-histidine tag, the69-87 sequence of GPMBP, and the C-terminal 118 aa fragment of IL-16.Primer design was based on the mouse IL-16 sequence. IL-16 cDNA wassynthesized in the presence of the 7H01 downstream primer. An overlapextension reaction included the 3F09 upstream primer and the 3F12 primerto generate the N-terminal HBM-7his-NAg construct together with the 3F11primer and the downstream 7H01 primer to amplify the IL16 DNA. Overlapof the constructs resulted in the resolution of an amplification productencoding the full length fusion protein. The IL16-S construct wasgenerated by a similar overlap extension - PCR strategy. The NAgIL16-SpFastbac1 plasmid was used as a template, and primers (6H08 and 7H11)were designed to delete the sequence encoding the GP69-87 NAg. The 3E06upstream primer and the 6H08 primer were used to generate an N-terminalHBM-7his construct as an extension product, and in the same reactiontube, the 7H11 primer and the 7H01 primer were used to amplify the IL-16sequence from the plasmid. Due to overlap between these two constructs,the final amplification product resolved as a contiguous HBMss-7his-IL16construct flanked by Spe I and Kpn I restriction endonuclease sites. Togenerate the NAgIL16-L construct, the NAgIL16-S pFastbac1 plasmid wasused as template for a whole plasmid PCR in the presence of the 7A01 and1F07 primers. 7A01 was the mutagenic primer containing a 2-nucleotidesubstitution (TTG substituted in place of for TCA) in the terminal 5′codon. This primer and the 1F07 primer were overlapping primers thatgenerated linearized copies of the plasmid.

TABLE 4 Vaccination with the IFNβ/NAg fusion protein elicits protectionagainst EAE. Mean Treatment of Incidence of Mean cumulative maximal Meanday of rats EAE score ± sd EAE ± sd onset ± sd Saline alone 13 of 1310.5 ± 3.0  2.9 ± 0.3  9.6 ± 1.5 GP68-89 15 of 15 8.6 ± 3.3 2.7 ± 0.610.7 ± 1.1 IFNβ.4 9 of 9 3.4 ± 2.7 1.4 ± 0.9 10.6 ± 0.5 The meancumulative scores and the mean maximal EAE of rats pretreated withIFNβNAg (IFNβ.4) was significantly different from those treated withsaline (p < 0.001) or GP68-89 (p < 0.001) (ANOVA, Tukey-Kramer MultipleComparisons Test).

TABLE 5 Vaccination with the IL2NAg fusion protein protected againstEAE. Median Median Mean # days Incidence cumulative maximal Mean dayIncidence of with severe Exp. # Treatment^(a) of EAE score^(b)intensity^(b) of onset^(b) severe EAE^(c) EAE^(d) 1 Saline alone 6 of 69.5 3.0 11.5 ± 1.5 6 of 6 4.7 ± 1.5 IL2.7-saline 3 of 4 1.0 0.3 15.3 ±1.5 0 of 4 0.0 2 Saline alone 6 of 6 11.1 3.0  8.7 ± 0.8 5 of 6 3.3 ±1.8 IL2.7-saline 5 of 6 1.1 0.4 12.8 ± 1.3 2 of 6 1.0 ± 1.6 IL2.7-alum 1of 6 0.0 0.0  8.0 ± 0.0 0 of 6 0.0 1&2 Saline alone 12 of 12 10.4 3.010.1 ± 1.9 11 of 12 4.0 ± 1.7 IL2NAg-  8 of 10 1.0 0.3 13.8 ± 1.8  2 of10 0.6 ± 1.4 saline IL2NAg- 1 of 6 0.0 0.0 8.0 0 of 6 0.0 alum ^(a)Datawere pooled from two independent experiments. In experiment #1, ratswere pretreated with saline or 0.5 nmole IL2.7 (IL2NAg) on days −60,−42, and −20 and were challenged with 50 ug GPMBP in CFA on day 0. Inexperiment #2, rats were pretreated with saline or 1 nmole IL2.7 (insaline or alum) on days −35, −21 and −7 and were challenged with 25 ugGPMBP in CFA on day 0. ^(b)Combined experiments: Median cumulativescores and median maximal intensity scores of rats pretreated withIL2NAg/saline (p < 0.001) or IL2NAg/alum (p < 0.001) were significantlyless than the respective scores for rats treated with saline (two-waynonparametric ANOVA on ranks, Bonferroni Post Hoc Test). The mean day ofonset of rats treated with IL2NAg in saline was significantly delayedcompared to that for rats pretreated with saline (unpaired t test, p =0.0004). ^(c)Combined experiments: Rats that exhibited ataxia (A), earlyparesis (EP), or full hindlimb paralysis (P) were scored as positive forsevere EAE. Incidence of severe EAE in rats pretreated with IL2NAg/alum(p = 0.0004) or with IL2NAg/saline (p = 0.0015) was significantly lessthan the respective incidence in rats pretreated with saline (pair-wisecomparisons by Fisher's Exact Test). ^(d)Each rat was scored for thetotal number of days that the rat exhibited severe EAE. The mean numberof days that IL2NAg/alum-pretreated rats (p < 0.001) andIL2NAg/saline-treated rats (p < 0.001) exhibited severe EAE wassignificantly less than that for saline pre-treated rats (two-wayparametric ANOVA, Bonferroni Post Hoc Test).

TABLE 6 Covalent tethering of IL-2 and NAg was necessary for tolerogenicactivity. Mean # of Median Median Incidence days with Incidencecumulative maximal Mean day of severe severe Exp. # Treatment^(a) of EAEscore^(b) intensity^(b) of onset^(c) EAE^(d) EAE^(d) 1 Saline alone 5 of5 9.5 2.0 8.2 ± 0.8 5 of 5 4.0 ± 1.0 GP69-88 5 of 5 9.8 3.0 8.8 ± 2.2 5of 5 3.8 ± 0.8 IL2 5 of 5 6.3 2.0 8.8 ± 1.3 5 of 5 4.4 ± 1.1 IL2 & GP69-4 of 4 10.8 2.5 8.8 ± 1.0 4 of 4 4.3 ± 1.0 88 IL2Ekdel 4 of 4 3.6 1.310.0 ± 0.8  2 of 4 1.5 ± 1.7 2 Saline alone 7 of 7 9.3 3.0 11.0 ± 1.2  7of 7 3.3 ± 1.0 GP69-88 7 of 7 8.5 3.0 10.7 ± 1.0  7 of 7 3.3 ± 0.5 IL2 8of 8 11.3 3.0 9.8 ± 0.9 8 of 8 3.5 ± 0.5 IL2 & GP69- 6 of 6 12.0 3.011.3 ± 0.8  6 of 6 3.3 ± 0.8 88 IL2Ekdel 8 of 8 4.6 2.0 12.5 ± 0.5  7 of8 2.0 ± 1.2 1 & 2 Saline alone 12 of 12 9.4 3.0 9.8 ± 1.7 12 of 12 3.6 ±1.0 NAg 12 of 12 9.0 3.0 9.9 ± 1.8 12 of 12 3.5 ± 0.7 IL2 13 of 13 9.33.0 9.4 ± 1.1 13 of 13 3.8 ± 0.9 IL2 & NAg 10 of 10 12.0 3.0 10.3 ± 1.6 10 of 10 3.7 ± 0.9 IL2NAg 12 of 12 4.3 2.0 11.7 ± 1.4   9 of 12 1.8 ±1.3 ^(a)Rats were pretreated with saline, 1 nmole of IL2Ekdel (IL2NAg),1 nmole GP69-88 (NAg), 1 nmole of IL2 (without NAg), or the combinationof GP69-88 and IL2. Rats (IL2 & GP69-88) were treated with separateinjections of 1 nmole IL2 and 1 nmole GP69-88 at a distance of <0.5 cmapart near the base of the tail. In experiment #1, rats were pretreatedon days −27, −20, and −13 and were challenged with 50 ug GPMBP in CFA onday 0. In experiment #2, rats were pretreated on days −35, −21, and −7and were challenged with 50 ug DHFR-NAg in CFA on day 0. ^(b)Combinedexperiments: The median cumulative score (p < 0.002 all comparisons) andthe median maximal score (p < 0.02 all comparisons) of rats pretreatedwith IL2NAg was significantly less than the respective scores of ratstreated with saline, NAg, IL-2 alone, or the combination of IL2 and NAg(two-way nonparametric ANOVA on ranks; Bonferroni Post Hoc Test).^(c)Combined experiments: The mean day of onset of rats pretreated withIL2NAg was significantly delayed compared to the respective means ofrats treated with either saline (p = 0.049) or IL-2 alone (p = 0.005)(two-way parametric ANOVA; Bonferroni Post Hoc Test). ^(d)Combinedexperiments: Rats that exhibited ataxia (A), early paresis (EP), or fullhindlimb paralysis (P) were scored as positive for severe EAE. Shown isthe incidence of severe EAE together with the mean number of days eachgroup exhibited severe EAE. The mean number of days that IL2NAg-treatedrats were severely afflicted with EAE was significantly less than therespective means for groups pretreated with saline, NAg, IL2, or thecombination of IL2 & NAg (p ≲ 0.001 for all comparisons) (two-wayparametric ANOVA; Bonferroni Post Hoc Test).

TABLE 7 IL2NAg was a more effective tolerogen than the IL4NAg fusionprotein. Mean # of days Mean Pooled Median Median Incidence withIncidence day of treatment cumulative maximal of severe severeTreatment^(a) of EAE onset^(b) groups^(c) score^(c) intensity^(c)EAE^(d) EAE^(e) Saline alone 15 of 15 10.1 ± 1.5 Saline 2.5 1.0  8 of 15(53% 1.6 ± 1.5 GPMBP/saline 3 of 4 12.3 ± 2.5 GPMBP 2.5 1.0  5 of 9 (56%1.2 ± 1.3 GPMBP/alum 4 of 5  7.0 ± 1.4 IL4.4/saline  9 of 10 11.0 ± 3.3IL4NAg 3.1 1.0 11 of 16 (69% 1.6 ± 1.2 IL4.4/alum 6 of 6  7.0 ± 0.6IL2.7/saline 2 of 5 16.5 ± 2.1 IL2NAg 0.3 0.1 0 of 10 (0% 0.0 ± 0.0IL2.7/alum 3 of 5  7.3 ± 1.5 ^(a)Rats were given subcutaneous injectionsof IL4.4 (IL4NAg), IL2.7 (IL2NAg), or GPMBP at a dose of 1 nmole on days−42, −28, and −14, and then were challenged with 25 ug GPMBP in CFA onday 0. ^(b)The mean day of onset of rats treated with IL2NAg in salinewas significantly delayed compared to that for rats treated with IL4NAgin saline (p = 0.0364) (Mann-Whitney U Test). To assess the effects ofthe alum adjuvant on the mean day of onset, pooled data for theGPMBP/saline, IL4NAg/saline, and IL2NAg/saline groups (n = 14) revealeda significant delay compared to that for pooled data of the GPMBP/alum,IL4NAg/alum, and IL2NAg/alum groups (n = 13) (p = 0.0002). Unpairedt-tests were used to confirm these differences: (GPMBP/saline vsGPMBP/alum; p = 0.0153), (IL4NAg/saline vs IL4NAg alum; p = 0.0128),(IL2NAg/saline vs IL2NAg alum; p = 0.0105). ^(c)Because the mediancumulative score and median maximal intensity were not affected by thesaline or alum adjuvant despite differences in mean day of onset, thedata for each respective protein injected in saline or alum were pooledfor statistical analysis of disease intensity. The median cumulativescore and the median maximal intensity score for rats injected withIL2NAg (pooled saline & alum groups) was significantly less than therespective medians of the control group (saline only; p < 0.01), thepooled GPMBP group (p < 0.05), and the pooled IL4NAg group (p < 0.001)(Kruskal-Wallis nonparametric ANOVA on ranks; Dunn's Multiple ComparisonTest). ^(d)Rats that exhibited ataxia (A), early paresis (EP), or fullhindlimb paralysis (P) were scored as positive for severe EAE. Theincidence of severe EAE in rats treated with IL2NAg was significantlyless than the respective incidences of rats treated with saline (p =0.0077), GPMBP (p = 0.0108), IL4NAg (p = 0.0007) by Fisher's Exact Test.These differences were confirmed for groups possessing a sufficient n(saline, IL4NAg, and IL2NAg) by the Chi-Squared Test for Independence.^(e)Each rat was scored for the total number of days that the ratexhibited severe EAE. Shown are the mean and standard deviation for eachpooled group (saline & alum). The mean number of days thatIL2NAg-treated rats were severely afflicted with EAE was significantlyless than the respective means of groups pretreated with saline (p <0.05), GPMBP (p < 0.05), or IL4NAg (p < 0.01) (ANOVA, Tukey-KramerMultiple Comparisons Test).

TABLE 8 Administration of IL2NAg after encephalitogenic sensitizationalso attenuated EAE. Mean # Median Median Mean Incidence days withIncidence cumulative maximal day of of severe severe Exp. #Treatment^(a) of EAE score^(b) intensity^(b) onset^(c) EAE^(d) EAE^(d) 1GP69-88 8 of 8 11.3 3.0 10.9 ± 1.4 8 of 8 3.1 ± 0.6 IL4Ekdel 6 of 6 7.63.0 12.8 ± 1.2 6 of 6 2.8 ± 0.4 IL2Ekdel 6 of 6 3.8 2.0 13.5 ± 0.8 5 of6 1.8 ± 1.0 2 GP69-88 8 of 8 7.5 3.0  9.9 ± 1.1 8 of 8 2.6 ± 0.9IL4Ekdel 9 of 9 6.3 2.0 12.4 ± 1.3 7 of 9 3.0 ± 1.3 IL2Ekdel 8 of 9 1.01.0 12.5 ± 2.6 3 of 9 1.1 ± 1.2 1 & 2 NAg 16 of 16 9.4 3.0 10.4 ± 1.3 16of 16 2.9 ± 0.8 IL4NAg 15 of 15 7.3 3.0 12.6 ± 1.2 14 of 15 2.9 ± 1.0IL2NAg 14 of 15 3.3 2.0 12.9 ± 2.0 10 of 15 1.4 ± 1.1 ^(a)Rats weresensitized with 50 ug DHFR-NAg in CFA on day 0. Rats were thensubcutaneously injected with 1 nmole of GP69-88 (NAg), IL4NAg(IL4Ekdel), or IL2NAg (IL2Ekdel) in saline on days 5, 7, and 9(experiment #1) or on days 5, 7, 9, and 11 (experiment #2). ^(b)Combinedexperiments: The median cumulative score and the median maximalintensity of rats treated with IL2NAg was significantly less than therespective medians for rats treated with either IL4NAg or NAg (p < 0.001for all comparisons) (two-way nonparametric ANOVA on ranks, BonferroniPost Hoc Test). ^(c)Combined experiments: The mean day of onset ofgroups treated with IL2NAg or IL4NAg was significantly delayed comparedto rats treated with NAg (p ≦ 0.001 for both comparisons) (two-wayparametric ANOVA; Bonferroni Post Hoc Test). ^(d)Combined experiments:Rats that exhibited ataxia (A), early paresis (EP), or full hindlimbparalysis (P) were scored as positive for severe EAE. The mean number ofdays that IL2NAg-treated rats were severely afflicted with EAE wassignificantly less than the respective means for groups treated withIL4NAg or NAg (p < 0.001 for each comparison) (two-way parametric ANOVA;Bonferroni Post Hoc Test).

TABLE 9 IL-16NAg and IL-2NAg vaccination protect against the subsequentactive induction of EAE. Mean Mean Mean day of Incidence cumulativemaximal onset^(d) ± Exp.^(a) Treatment of EAE score^(b) ± sd EAE^(c) ±sd sd 1 Saline 10 of 10 9.4 ± 3.1 2.5 ± 0.7 10.6 ± 1.3 alone GPMBP 7 of7 8.3 ± 4.1 2.6 ± 0.8 10.4 ± 0.8 IL2.7 7 of 8 4.4 ± 3.3 1.8 ± 1.0 12.1 ±1.2 NAgIL16- 7 of 8 3.2 ± 2.5 1.5 ± 0.9 12.6 ± 1.0 S 2 Saline 6 of 6 9.2± 0.7 3.0 ± 0.0 10.0 ± 1.5 alone GP69-88 7 of 7 8.5 ± 1.4 2.9 ± 0.4 10.4± 0.5 IL2EKdel 9 of 9 5.1 ± 2.6 2.1 ± 0.7 12.2 ± 0.7 NAgIL16- 9 of 9 2.4± 1.2 1.1 ± 0.7 12.2 ± 0.4 L Combined Saline 16 of 16 9.3 ± 2.4 2.7 ±0.6 10.4 ± 1.4 alone NAg 14 of 14 8.4 ± 3.0 2.7 ± 0.6 10.4 ± 0.6 IL2NAg16 of 17 4.8 ± 2.9 1.9 ± 0.8 12.2 ± 0.9 NAgIL16 16 of 17 2.8 ± 1.9 1.3 ±0.8 12.4 ± 0.7 ^(a)In experiment #1, rats were pretreated with1 nmoleGPMBP, IL2.7, or NAgIL16.1 on days −31, −17, −7 and were challenged with50 μg GPMBP in CFA on day 0. In experiment #2, rats were pretreated 1nmole synthetic peptide GP69-88, IL2Ekdel, or rat NAgIL16 on days −21,−14, −7 and were challenged with 50 μg DHFR/NAg in CFA on day 0.^(b)Combined experiments: Mean cumulative scores of rats pretreated withIL16NAg or IL2NAg were significantly different from those treated withsaline (p < 0.001) or NAg (p < 0.001 and 0.01 respectively). The meanmaximal intensity of rats pretreated with IL16NAg was significantlydifferent from those treated with IL2NAg (p < 0.05), NAg (p < 0.001), orsaline (p < 0.001). The mean maximal intensity of rats pretreated withIL2NAg was significantly different from those treated with NAg (p <0.05) or saline (p < 0.05). The mean day of onset of rats pretreatedwith IL16NAg or IL2NAg was significantly different from those treatedwith NAg or saline (p < 0.001).

TABLE 10 Inhibitory Activity of IL10.6, IL-13.6 and IL1RA/NAg fusionproteins. Mean cumulative Mean maximal Mean day of Exp. TreatmentIncidence of EAE score ± sd intensity ± sd onset ± sd 1 Saline 7 of 79.4 ± 4.4 2.9 ± 0.7 10.7 ± 1.0 GP69-88 8 of 8 7.3 ± 3.7 2.4 ± 1.3 11.1 ±2.2 IL10.6 8 of 8 7.3 ± 3.8 2.3 ± 1.0 11.3 ± 1.3 2 Saline 17 of 17 9.5 ±2.6 2.7 ± 0.5 10.6 ± 1.2 GPMBP 20 of 20 7.0 ± 3.4 2.4 ± 0.9 10.9 ± 0.9IL13.6 15 of 15 5.2 ± 2.9^(a) 2.0 ± 1.1 11.6 ± 0.7 IL1RA/NAg4 13 of 134.8 ± 2.9^(a) 1.7 ± 0.9^(b) 12.2 ± 1.2^(c) ^(a)The mean cumulativescores of rats pretreated with IL13NAg or IL1RA/NAg significantlydiffered from those treated with saline (p < 0.001). ^(b)The meanmaximal intensity of rats pretreated with IL1RA/NAg4 was significantlydifferent from those treated with saline (p < 0.01). ^(c)The mean day ofonset of rats pretreated with IL1RA/NAg4 was significantly differentfrom those treated with NAg or (p < 0.01) or saline (p < 0.001).

TABLE 11 Covalent tethering of IL-16 and NAg was involved in tolerogenicactivity. Mean Treatment of Incidence of cumulative Mean maximal Meanday of Incidence of rats^(a) EAE score ± sd^(b) intensity ± sd onset ±sd relapse Saline alone 6 of 6 10.5 ± 4.2  2.8 ± 0.4 10.3 ± 0.5 4 of 6(33%) NAgIL16-L 9 of 9 2.2 ± 1.7 0.8 ± 0.7 13.2 ± 0.8 0 of 9 (0%) GP69-88 7 of 7 9.0 ± 2.0 2.7 ± 0.5 10.3 ± 0.5 2 of 7 (29%) IL16-S 8 of 812.5 ± 2.6  3.0 ± 0.0  9.9 ± 1.0 5 of 8 (63%) IL16-S & 7 of 7 9.0 ± 2.12.9 ± 0.4 10.3 ± 0.5 3 of 7 (43%) GP69-88 ^(a)Rats were pretreated withsaline, 1 nmole of NAgIL16-L, 1 nmole GP69-88, 1 nmole of IL16-S (noNAg), or the combination of GP69-88 and IL16-S. Rats (5^(th) row) weretreated with separate injections of 1 nmole IL16-S and 1 nmole GP69-88at a distance of <0.5 cm apart near the base of the tail. Rats werepretreated on days −21, −14, and −7 and were challenged with 50 μgDHFR-NAg in CFA on day 0. ^(b)The mean cumulative score, the meanmaximal intensity, and the mean day of onset of rats pretreated withNAgIL16-L was significantly different from those treated with saline,GP69-88, IL16-S alone, or the combination of IL16-S and GP69-88 asseparate molecules (p < 0.001). (ANOVA, Tukey-Kramer MultipleComparisons Test).

TABLE 12 High-dose, systemic treatment with NAgIL16-L inhibited theeffector phase of EAE. Mean cum. Mean max. Mean day of Incidence ofTreatment^(a) Incidence Score ± sd intensity ± sd onset ± sd relapseGP69-88 4 of 4 8.9 ± 2.6 3.0 ± 0.0 10.5 ± 0.6 2 of 4 (50%) NagIL16-L^(b)5 of 5 1.5 ± 0.9 0.5 ± 0.3 12.5 ± 1.0 0 of 5 (0%)  ^(a)Rats werechallenged with 50 μg DHFR-NAg in CFA on day 0. On day 8, rats weretreated with 5 nmoles of GP69-88 or NagIL16-L intravenously. On day 12,rats were treated with 5 nmoles of GP69-88 or NagIL16-L byintraperitoneal injection. Injection of GP69-88 or NagIL16-L via theseroutes did not cause any notable adverse reaction. In the IL16-Ltreatment group, one rat exhibited initial signs of EAE (0.25) beforeadministration of the first treatment. None of the other rats exhibitedEAE at this timepoint (day 8). The day of onset for this rat wasexcluded from the calculation of the mean day of onset for the NagIL16-Ltreatment group. ^(b)Mean cumulative score, the mean maximal intensity,and the mean day of onset of rats treated with NagIL16-L weresignificantly different from rats treated with GP69-88 (unpaired t test,p < 0.0005, p < 0.0001, and p < 0.0135, respectively).

TABLE 13 Subcutaneous IL2Ekdel and NagIL16-L injected on days 5, 7, & 9attenuated EAE. Mean cum. Mean max. Mean day of Incidence ofTreatment^(a) Incidence score^(b) ± sd intensity ± sd onset^(b) ± sdrelapse Saline 8 of 8 10.3 ± 4.7  2.5 ± 0.9 11.0 ± 1.1 6 of 8 (75%)GP69-88 8 of 8 10.5 ± 2.8  3.0 ± 0.0 10.9 ± 1.4 6 of 8 (75%) IL4Ekdel 6of 6 7.6 ± 1.1 3.0 ± 0.0 12.8 ± 1.2 2 of 6 (33%) IL10.6 6 of 6 6.5 ± 1.02.8 ± 0.4 12.5 ± 0.8 0 of 6 (0%)  IL13.6 6 of 6 6.4 ± 4.0 2.3 ± 1.2 12.3± 1.5 2 of 6 (33%) IL1RA/NAg4 6 of 6 5.5 ± 2.0 2.4 ± 1.0 12.8 ± 0.4 0 of6 (0%)  NAgIL16-L 6 of 6 4.7 ± 1.8 2.1 ± 0.9 12.5 ± 1.0 0 of 6 (0%) IL2Ekdel 6 of 6 4.3 ± 2.4 2.0 ± 1.0 13.5 ± 0.8 0 of 6 (0%)  ^(a)Ratswere challenged with 50 μg DHFR-NAg in CFA on day 0. On days 5, 7, and9, rats were treated with 1 nmole of GP69-88 or 1 nmole of thedesignated cytokine/NAg fusion protein by subcutaneous injection.^(b)Mean cumulative scores of rats treated with IL2Ekdel (p < 0.01) orwith NAgIL16-L (p < 0.05) were significantly different from meancumulative scores of rats treated with GP69-88 or saline. The meancumulative score of rats treated with IL1RA/NAg4 was significantlydifferent for that for rats treated with GP69-88 (p < 0.05). Mean day ofonset of rats treated with IL1RA/NAg4 (p < 0.05), IL2Ekdel (p < 0.01),or IL4Ekdel (p < 0.05) were significantly different from the mean day ofonset of rats treated with GP69-88. Mean day of onset of rats treatedwith IL2Ekdel (p < 0.01)was significantly different from the mean day ofonset of rats treated with saline. (ANOVA; Tukey-Kramer MultipleComparisons Test)

TABLE 14 The NAgIL16 vaccine was effective when delivered duringsensitization. Mean cumulative Mean max. Mean day Incidence of Exp.^(a)Treatment^(a) Incidence score EAE of onset relapse 1^(b) Saline 5 of 511.8 ± 2.6  3.0 ± 0.0 11.2 ± 1.3 IL1RA/NAg4 5 of 5 5.3 ± 3.8 2.0 ± 1.013.0 ± 1.2 NAgIL16 5 of 5 5.3 ± 4.4 1.7 ± 1.3 12.0 ± 1.9 2^(c) Saline 10of 10 11.6 ± 7.3  3.0 ± 0.0  9.6 ± 0.8 7 of 8 (88%) GP69-88 8 of 8 11.2± 2.7  3.0 ± 0.0 10.4 ± 0.7 5 of 8 (63%) IL1RA/NAg4 6 of 6 8.3 ± 3.9 2.5± 0.8 11.7 ± 1.0 3 of 6 (50%) NAgIL16 8 of 9 4.0 ± 3.1 1.9 ± 1.2 12.3 ±0.5 1 of 8 (13%) ^(a)Rats were challenged with 50 μg GPMBP (experiment#1) or DHFR-NAg (experiment #2) in CFA on day 0. On days −1, 3, and 7,rats were treated with saline, GP69-88, or the designated cytokine/NAgfusion protein by subcutaneous injection. Either 1 nmole or 2 nmoledoses per injection were administered in experiments 1 and 2,respectively. ^(b)In experiment 1, mean cumulative score of rats treatedwith NAgIL16 or IL1RA/NAg4 was significantly different from thosetreated with saline (p < 0.05). ^(c)In experiment 2, mean cumulativescore and the mean maximal intensity of rats treated with NAgIL16 wassignificantly different from those treated with saline (p < 0.01) orGP69-88 (p < 0.05). The mean day of onset of rats treated with NAgIL16was significantly different from those treated with saline or GP69-88 (p< 0.001). The mean day of onset of rats treated with IL1RA/NAg4 wassignificantly different from those treated with saline (p < 0.001) orGP69-88 (p < 0.05).

TABLE 15 NAgIL16-L treatment causes the antigen-specific inhibition ofthe encephalitogenic effector phase. Mean Mean Mean number cumulativemaximal Mean day Incidence of of days afflicted Incidence score^(b) ±intensity^(c) ± of onset ± severe EAE with severe Treatment^(a) of EAEsd sd sd (EP & P)^(d) EAE^(e) NAgIL16L 6 of 8 2.5 ± 2.5 1.0 ± 1.0 10.5 ±0.5 2 of 8 (25%)  0.4 ± 0.7 Both 8 of 8 6.8 ± 2.3 2.6 ± 0.5 10.1 ± 0.8 8of 8 (100%) 2.0 ± 0.8 GP69-88 8 of 8 7.4 ± 2.1 2.9 ± 0.4  9.5 ± 1.2 8 of8 (100%) 2.4 ± 1.2 IL-16 8 of 8 7.3 ± 1.1 3.0 ± 0.0 10.0 ± 0.9 8 of 8(100%) 2.1 ± 0.4 ^(a)Rats were challenged with 50 μg DHFR-NAg in CFA onday 0 and then were treated on day 8 with intravenous injections of 5nmoles NAgIL16-L (1^(st) row), a combined solution of 5 nmoles IL-16 & 5nmoles GP69-88 (2^(nd) row), 5 nmoles GP69-88 (3^(rd) row), or 5 nmolesIL-16 (4^(th) row). These same injections (5 nmoles) for each group wererepeated by intraperitoneal injection on day 12. ^(b)Parametric ANOVA(Tukey-Kramer Multiple Comparison's Test) revealed that the meancumulative score of rats treated with NAgIL16 significantly differedfrom the respective scores for groups treated with IL-16 & GP69-88 (p <0.01), GP69-88 (p < 0.001), or IL-16 (p < 0.001). ^(c)NonparametricKruskal-Wallis ANOVA (Dunn's Multiple Comparisons Test) revealed thatthe mean maximal intensity score of rats treated with NAgIL16significantly differed from those for groups treated with IL-16 &GP69-88 (p < 0.05), GP69-88 (p < 0.01), or IL-16 (p < 0.001). ^(d)Ratsthat exhibited early paresis (EP) or full hindlimb paralysis (P) werescored as positive for severe EAE. The incidence of severe EAE in ratstreated with NAgIL16L significantly differed from that for rats treatedwith the combination of IL-16 & GP69-88 (p = 0.007) by Fisher's ExactTest. ^(e)Each rat was scored for the total number of days that the ratexhibited severe EAE (EP or P). Shown are the mean and standarddeviation for each group. Nonparametric Kruskal-Wallis ANOVA (Dunn'sMultiple Comparisons Test) revealed that the mean number of days thatNAgIL16L-treated rats were severely afflicted with EAE significantlydiffered from the respective means of groups that were treated with thecombination with IL-16 & GP69-88 (p < 0.05), GP69-88 (p < 0.01), orIL-16 (p < 0.01).

TABLE 16 A pre- and post-challenge NAgIL16 treatment regimen inhibitsdisease incidence. Mean number of days^(e) Incidence afflicted with:Mean Mean of severe mild Incidence cumulative maximal Mean day of EAEEAE severe Treatment^(a) of EAE^(b) score^(c) intensity^(c) onset^(d)(A, EP, P)^(e) (dLT-LT) EAE (A-P) NAgIL16L 2 of 9 0.3 ± 0.8 0.3 ± 0.714.0 ± 2.8 1 of 9 0.3 ± 0.7 0.1 ± 0.3 GP69-88 9 of 9 6.9 ± 3.3 2.8 ± 0.710.3 ± 1.4 9 of 9 2.4 ± 1.0 2.9 ± 1.4 Saline 9 of 9 8.5 ± 2.1 2.7 ± 0.5 8.8 ± 0.7 9 of 9 2.6 ± 1.0 3.8 ± 0.8 ^(a)Rats were pretreated with 2nmoles NAgIL16L (1^(st) row), 2 nmoles GP69-88 (2^(nd) row), or saline(3^(rd) row) on days −21, −14, and −7. Rats were challenged with 50 ugDHFR-NAg in CFA on day 0 and then were treated on days 8 and 11 withintraperitoneal injections of 5 nmoles of the respective antigen orsaline. ^(b)The incidence of EAE of rats treated with NAgIL16L wassignificantly less compared to the incidence of EAE in rats treated withGP69-88 (p = 0.0023) (Fisher's Exact Test). ^(c)The mean cumulativescore of rats treated with NAgIL16 was significantly reduced compared tothe respective scores for rats treated with GP69-88 (p < 0.01) or saline(p < 0.001). The mean maximal intensity of rats treated with NAgIL16 wassignificantly reduced compared to the respective scores for rats treatedwith GP69-88 (p < 0.001) or saline (p < 0.01) (NonparametricKruskal-Wallis ANOVA; Dunn's Multiple Comparisons Test). ^(d)The meanday of onset of rats treated with NAgIL16 was significantly delayedcompared to the respective disease onset for rats treated with GP69-88(p < 0.01) or saline (p < 0.001) (parametric ANOVA; Tukey-KramerMultiple Comparisons Test). ^(e)Rats that exhibited distal limp tail(dLT) or limp tail (LT) were scored as positive for mild EAE, and ratsthat exhibited ataxia (A), early paresis (EP), or full hindlimbparalysis (P) were scored as positive for severe EAE. The incidence ofsevere EAE in rats treated with NAgIL16L was significantly reducedcompared to that for rats treated with GP69-88 (p = 0.0004) (Fisher'sExact Test). The mean number of days that NAgIL16L-treated rats wereeither mildly or severely afflicted with EAE was significantly reducedcompared to the respective means of groups that were treated withGP69-88 (p < 0.001) or saline (p < 0.001) (parametric ANOVA;Tukey-Kramer Multiple Comparisons Test).

TABLE 17 Examination of combinations of vaccines for synergy forinduction of tolerance. Treatment groups (n = 10)  1. NAgIL16-L (1xdose)  2. IFNβ.4 (1x dose)  3. IL2.7 (1x dose)  4. GP68-89 (1x dose)  5.NAgIL16-L (2x dose)  6. IFNβ.4 (2x dose)  7. IL2.7 (2x dose)  8. GP68-89(2x dose)  9. NAgIL16-L plus IL2.7 10. NAgIL16-L plus IFN□.4 11. IL2.7plus IFNβ.4 12. Saline

TABLE 18 Tolerance elicited by cytokine/NAg vaccines isantigen-specific. Tolerance induction Challenge of rats with:Outcome: 1. NAgIL16-L DHFR-NAg in CFA Resistance to EAE 2. IL2.7 P0peptide 180-199/CFA Full susceptibility to EAN 3. IFNβ.4 P0 peptide56-71/CFA Full susceptibility to EAN 4. Optimal combinations. P2 peptide53-81/CFA Full susceptibility to EAN 5. GP68-89 6. Saline

TABLE 19 NAgIL16 treatment inhibits progression of EAE when administeredafter disease onset Mean cumulative Median Mean Median score (2.5cumulative Mean Median Incidence Incidence cumulative cumulative dayscore (2.5 day intensity intensity severe EAE Exp. Treatment^(a) of EAEscore score window) window) score score (EP & P) 1 NAg 7 of 7 13.3 ±7.0  12.5 4.9 ± 2.5 4.5 2.1 ± 1.0 2.0 5 of 7 NAgIL16 8 of 8 6.7 ± 5.44.5 1.2 ± 0.7 1.0 1.0 ± 0.7 1.0 2 of 8 2 NAg 7 of 7 9.1 ± 1.6 8.3 3.6 ±1.8 3.8 2.0 ± 0.8 2.0 5 of 7 NAgIL16 7 of 7 3.5 ± 1.5 3.3 1.0 ± 0.5 0.80.9 ± 0.6 0.5 1 of 7 1 & 2 NAg 14 of 14 11.2 ± 5.4  10.0 4.3 ± 2.2 4.42.0 ± 0.9 2.0 10 of 1  NAgIL16^(b) 15 of 15 5.2 ± 4.2 3.8 1.1 ± 0.6 0.81.0 ± 0.6 1.0 3 of 1 ^(a)Rats were challenged with 50 ug DHFR-NAg in CFAon day 0. Rats were randomly assigned to one of two treatment groupsbased on expression of distal limp tail (0.25) or limp tail (0.5)immediately before the first treatment. In experiment 1 (NAg group;0.25, n = 2; 0.5, n = 5 and NAgIL16 group; 0.25, n = 2; 0.5, n = 6),treatments were administered on day 10 (5 nmoles i.v.) and day 11 (5nmoles i.p.). In experiment 2 (NAg group; 0.25, n = 2; 0.5, n = 5 andNAgIL16 group; 0.25, n = 1; 0.5, n = 6), NAg and NAgIL16 wereadministered on day 11 (5 nmoles i.v.), day 12 (5 nmoles i.p.), and day14 (2 nmoles i.v.). In experiments 1 & 2, rats were scored onapproximate 12 hr intervals. ^(b)The mean and median cumulative score,the mean and median cumulative scores for a 2.5-day window following thesecond treatment (days 12-14.5 and days 13-15.5 for experiments 1 & 2respectively), and the mean and median maximal intensity scores forNAgIL16-treated rats were significantly less than those for NAg-treatedrats (p < 0.001, Independent Samples T-Test). Differences in medianvalues were assessed by comparison of ranked data. The incidence ofsevere EAE in NAgIL16-treated rats was significantly less than therespective incidence in NAg-treated rats (Fisher's Exact Test, p =0.0092).

TABLE 20 Covalent tethering of IL-2 and NAg involvement in EAEinhibition with IL2NAg treatment during onset of EAE. Median Median MeanIncidence Mean # days Incidence cumulative maximal day of of severe withsevere Treatment^(a) of EAE score^(b) intensity^(b) onset^(c) EAE^(d)EAE^(d) Saline alone 10 of 10 8.6 3.0 9.8 ± 0.6 10 of 10  3.0 ± 0.7 IL2& GP69-88 10 of 10 7.9 3.0 9.9 ± 0.9 9 of 10 3.0 ± 1.3 IL2NAg  8 of 100.6 0.3 11.5 ± 1.6  2 of 10 0.5 ± 1.1 ^(a)Rats were sensitized with 50ug DHFR-NAg in CFA on day 0. Rats were then injected with saline, 1nmole of IL2NAg (IL2Ekdel) in saline, or with separate injections of 1nmole IL2 and 1 nmole GP69-88 in saline s.c. at a distance of <0.5 cmapart near the base of the tail on days 5, 7, 9, 11, and 13. ^(b)Themedian cumulative score of rats treated with IL2NAg was significantlyless than the respective scores of rats treated with saline or thecombination of IL2 and GP69-88 (p < 0.01 or p < 0.05 respectively). Themedian maximal score (p < 0.01 all comparisons) of rats pretreated withIL2NAg was significantly less than the respective scores of rats treatedwith saline or the combination of IL2 and GP69-88 (Kruskal-Wallisnonparametric ANOVA on ranks; Dunn's Multiple Comparison Test). ^(c)Themean day of onset of rats pretreated with IL2NAg was significantlydelayed compared to the respective means of rats treated with eithersaline (p < 0.01) or the combination of IL-2 and GP69-88 (p < 0.05)(one-way parametric ANOVA; Tukey-Kramer Multiple Comparisons Test).^(d)Rats that exhibited ataxia (A), early paresis (EP), or full hindlimbparalysis (P) were scored as positive for severe EAE. The incidence ofsevere EAE in rats treated with IL2NAg was significantly less than theincidence for rats treated with the combination of IL2 and NAg (p =0.0055, Fisher's Exact Test). The mean number of days thatIL2NAg-treated rats were severely afflicted with EAE was significantlyless than the respective means for groups pretreated with saline or thecombination of IL2 & GP69-88 (p ≦ 0.001 for all comparisons) (one-wayparametric ANOVA; Tukey-Kramer Multiple Comparisons Test).

TABLE 21 The combination of NAgIL16 and IL2NAg strongly inhibits theeffector phase of EAE. Mean Median Mean Median Incidence Mean # daysIncidence cumulative cumulative maximal maximal Mean day of severe withsevere Treatment^(a) of EAE score score score score of onset EAE (A-P)EAE Control 7 of 7 8.5 ± 3.3 9.8 2.6 ± 0.8 3.0 9.4 ± 1.3 7 of 7 2.6 ±1.4 Cytokine/ 5 of 5 0.5 ± 0.3 0.5 0.2 ± 0.1 0.3 11.3 ± 3.4  0 of 5 0.0NAg

TABLE 22 NAgIL16 induces an active, dominant mechanism of tolerance.Incidence Mean # Mean Median Mean Median of days with Incidence ofcumulative cumulative maximal maximal severe severe Exp. Treatment^(a)EAE score score score score EAE EAE 1 control 9 of 9 9.1 ± 2.7 10.0 2.8± 0.4 3.0 9 of 9 2.8 ± 1.0 females NAg 4 of 4 7.2 ± 3.0 6.8 2.8 ± 0.53.0 4 of 4 2.3 ± 1.3 NAgIL16 4 of 4 3.9 ± 1.9 3.8 1.9 ± 1.0 2.0 3 of 41.0 ± 0.8 2 control 10 of 10 9.4 ± 2.9 8.5 2.8 ± 0.4 3.0 10 of 10 2.9 ±0.9 males NAg 6 of 6 12.5 ± 2.4  12.3 3.0 ± 0.0 3.0 6 of 6 3.8 ± 1.2NAgIL16 6 of 6 7.4 ± 4.7 7.6 2.1 ± 1.1 2.5 4 of 6 2.0 ± 1.7 1 & 2control 19 of 19 9.2 ± 2.8 9.5 2.8 ± 0.4 3.0 19 of 19 2.8 ± 0.9 NAg 10of 10 10.4 ± 3.7  11.0 2.9 ± 0.3 3.0 10 of 10 3.2 ± 1.4 NAgIL16^(b) 10of 10 6.0 ± 4.1 5.4 2.0 ± 1.0 2.0  7 of 10 1.6 ± 1.4 ^(a)Rats werepretreated with 4 nmoles of GP69-88 (NAg) or 4 nmoles NAgIL16 in salineon days −28, −21, and −14. On day −7, draining lymph node cells andsplenocytes were pooled from rats of each group and were injected (i.p.)into recipient rats (1:1 donor to recipient ratio). Seven days afteradoptive transfer, recipient rats were challenged on day 0 with 50 ug ofDHFR-NAg in CFA. ^(b)Combined experiments 1 & 2: Compared to recipientsof NAgIL16-treated donor cells, the mean (p = 0.009, p = 0.034) andmedian (p = 0.007, p = 0.066) cumulative score, the mean (p = 0.010, p =0.010) and median (p = 0.021, p = 0.049) maximal score, and the meannumber of days afflicted with severe EAE (EP & P) (p = 0.010, p = 0.024)were significantly less than the respective scores of recipients ofNAg-treated donor cells or untreated (control) rats, respectively.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

What is claimed is:
 1. A method of treating multiple sclerosiscomprising administering to a subject an effective amount of a first andat least a second fusion protein, wherein the first and the at leastsecond fusion protein each independently comprise: (a) anencephalitogenic determinant of an autoimmune antigen selected from thegroup consisting of myelin basic protein (MBP), proteolipid protein(PLP), myelin oligodendrocyte glycoprotein (MOG) and myelin-associatedoligodendrocytic basic protein; and (b) an anti-inflammatory cytokineselected from the group consisting of an interleukin or interleukinreceptor antagonist, wherein the anti-inflammatory cytokine of the firstfusion protein is interleukin-2 (IL-2) and the anti-inflammatorycytokine of the at least second fusion protein is interleukin-4 (IL-4),interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-16 (IL-16),interferon-beta (IFN-β) or transforming growth factor-beta (TGF-β) andthe interleukin receptor antagonist is interleukin-1RA (IL-1RA).
 2. Themethod of claim 1, wherein the autoimmune antigen is a myelin basicprotein.
 3. The method of claim 2, wherein the encephalitogenicdeterminant of the myelin basic protein comprises the amino acidsequence of SEQ ID NO:2.
 4. The method of claim 1, wherein theanti-inflammatory cytokine of the at least second fusion protein isinterleukin-4 (IL-4), or interleukin-16 (IL-16).
 5. The method of claim1, wherein the anti-inflammatory cytokine of the at least second fusionprotein is interleukin-16 (IL-16).
 6. The method of claim 1, wherein theanti-inflammatory cytokine of the at least second fusion protein isinterferon-beta (IFN-β).
 7. The method of claim 1, wherein theanti-inflammatory cytokine of the at least second fusion protein istransforming growth factor-beta (TGF-β).
 8. The method of claim 1,wherein the autoimmune antigen is a proteolipid protein.
 9. The methodof claim 1, wherein the autoimmune antigen is a myelin oligodendrocyteglycoprotein.
 10. A method of treating multiple sclerosis comprisingadministering to a subject an effective amount of a first and at least asecond fusion protein, wherein the first and the at least second fusionprotein each independently comprise: (a) an encephalitogenic determinantof an autoimmune antigen selected from the group consisting of myelinbasic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyteglycoprotein (MOG) and myelin-associated oligodendrocytic basic protein;and (b) an anti-inflammatory cytokine selected from the group consistingof an interleukin or interleukin receptor antagonist, wherein theinterleukin of the first fusion protein is interleukin-16 (IL-16) andthe interleukin of the at least second fusion protein is interleukin-2(IL-2).